Subsequent Plant Regeneration Research Articles

Gibberellic Acid + 2,4‐D Improves Seed Quality in Hordeum vulgare L. ×H. bulbosum L. Crosses

AbstractDoubled haploid production from crosses between barley (Hordeum vulgare L.) and H. bulbosum L. is a well established technique for inducing homozygosity. One problem which occurs using certain barley cultivars is premature seed degeneration. In this paper, a post‐pollination spray of gibberellic acid and 2,4‐dichlorophenoxyacetic acid that delays the onset of senescence is described. The technique improved the number of high quality seeds to more than 75% of the seed set. This improvement should result in increased survival of embryos for culture and subsequent plant regeneration.

Protein markers for anther culturability in barley

Two-dimensional electrophoresis of proteins from a recombinant population of anther culture-derived doubled haploid lines identified 4 loci or linkage groups showing a deviation from an expected 1∶1 segregation. It was hypothesized that these markers are linked to genes involved in the process of haploid plant production and that the deviation was due to a selection for alleles conferring higher anther culture response. To check this hypothesis, the anther culturability of 50 of the doubled haploid lines and their two inbred parents was assessed. It was found that 2 of the loci which had a distortion of segregation showed a significant effect on anther culture response, the most efficient allele being the most frequent in both loci. In addition, 2 more markers associated with anther culturability were found. One of the first mentioned 2 loci and one of the latter 2 were found to be linked to genes involved in both embryoid production and subsequent green plant regeneration. The remaining two were linked to genes involved only in green plant regeneration. Of the 4 favorable alleles 3 were inherited from one parent.

Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration

In vitro-grown apical meristems of wasabi (Wasabia japonica Matsumura) were successfully cryopreserved by vitrification. Excised apical meristems precultured on solidified M S medium containing 0.3M sucrose at 20°C for 1 day were loaded with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) for 10 min at 25°C prior to a plunge into liquid nitrogen. After rapid warming, the meristems were expelled into 2 ml of 1.2M sucrose for 20 min and then plated on solidified culture medium. Successfully vitrified and warmed meristems remained green after plating, resumed growth within 3 days, and directly developed shoots within two weeks. The average rate of normal shoot formation amounted to about 80 to 90% in the cryopreserved meristems. This method was successfully applied to three other cultivars of wasabi. This vitrification procedure promises to become a routine method for cryopreserving meristems of wasabi.

Somatic embryogenesis and organogenesis inGuizotia Abyssinica

Induction of somatic embryogenesis, shoot organogenesis, and subsequent plant regeneration in niger seem to be dependent on genotype, choice of explant, and composition of media growth regulators. Two distinct regeneration protocols have been developed for somatic embryogenesis and shoot organogenesis. Somatic embryogenesis was induced from epicotyls and cotyledonary explants (9 to 35%) (but not from hypocotyls and roots) in presence of 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxypropionic acid. These embryos matured in MS medium containing Kinetin plus naphthalene acetic acid (NAA), Kinetin plus Zeatin, and Kinetin plus abscisic acid (ABA). Matured embryos could be germinated on LS and MS basal media without hormones. Non-embryogenic callus initiated on Linsmaier and Skoog’s (LS) medium from cotyledons of six different genotypes produced shoots (9 to 32%) on Murashige and Skoog’s (MS) medium fortified with 6-benzylaminopurine (BAP, 0.5 mg · liter−1), and BAP (1 mg · liter−1) plus NAA (0.1 mg · liter−1). These shoots were rooted with 100% frequency by using indole-3-acetic acid or NAA and transferred successfully to the soil.

Somatic embryogenesis and plant regeneration in a woody species: the European Spindle Tree (Euonymus europaeus L.)

Somatic embryogenesis and subsequent plant regeneration of Euonymus europaeus L (European Spindle Tree) were obtained from square pieces of mature zygotic embryos with an intervening callus phase. Callus and somatic embryos were induced using a Murashige and Skoog's semi-solid basal medium supplemented with several combinations of auxins and cytokinins. The greatest number of somatic embryos was obtained with a continuous exposure to 22.8 μM indoleacetic acid and 0.046 μM kinetin. The frequency of somatic embryogenesis from zygotic embryos depends on the cold conservation time of seeds. The embryos frequently germinated on the same medium. Further development of somatic embryos into plantlets was achieved on a medium devoid of growth regulators.

Protoplasts to plants of Gentianaceae. Regeneration of lisianthus (Eustoma grandiflorum) is affected by calcium ion preconditioning, osmolality and pH of the culture media

A reliable method has been developed for regeneration of whole plants from isolated protoplasts of five cultivars of lisianthus,Eustoma grandiflorum (Griseb.) Schinners (Gentianaceae). Protoplasts were isolated from either cotyledons or leaves and cultured in agarose beads surrounded by liquid V-KM media containing 5.37 µM 1-naphthyleneacetic acid (NAA) and 2.28 µM zeatin. When microcalli were approximately 1 mm in diameter, the agarose beads were transferred to shoot regeneration media containing 0.1 µM indolebutyric acid (IBA) and 4.44 µM 6-benzylaminopurine (BAP). Shoots were produced from the calli during several sub-culture periods. Protoplast viability and the subsequent regeneration of plants were dependent on calcium levels and growth regulator presence in thein vitro seed germination media, on the osmolality of the protoplast purification solution, and osmolality increase and pH of the culture media. Shoots were rooted in Murashige & Skoog (1962) media containing 5.71 µM indole-3-acetic acid (IAA). Plantlets derived from protoplasts of five lisianthus cultivars (Fresh White, Hakusen, Miss Lilac, Fresh Purple and Doremi Wine Red) have been successfully transferred to the glasshouse.

Polyamine Metabolism Associated with Growth and Embryogenic Potential of Rice

Summary To determine whether polyamines are involved in somatic embryogenesis in a monocot, levels of polyamines were determined using HPLC during embryogenic callus induction and during cell-culture growth of rice ( Oryza sativa L.). Free putrescine accumulated ten-fold in cells during callus induction and growth using conditions optimized for subsequent plant regeneration. Free and conjugated pools of putrescine, spermidine, and spermine in cell suspension cultures exhibited a regular, proportional pattern of increase and decline during each subculture cycle. Polyamine biosynthesis inhibitors, difluoromethylarginine (DFMA) and difluoromethylornithine (DFMO), were used to determine the importance of de novo putrescine biosynthesis for explant dedifferentiation and growth of embryogenic rice callus. Callus was induced in the presence of 2,4-D and either of these inhibitors. However, DFMA (10 mM) suppressed cell suspension culture growth and plant regeneration from callus. These effects were reversible with exogenous agmatine. DFMO inhibited cell suspension culture growth only in the absence of exogenous 2,4-D and had no effect on plant regeneration from callus. A comparison of the morphogenesis of cell suspensions cultured in the presence of DFMA or DFMO revealed that these inhibitors differ in their impact on cell differentiation, proliferation of compact meristematic cell clusters, starch metabolism, and cell cluster friability. We conclude that polyamine metabolism via arginine decarboxylase influences the embryogenic potential of in vitro rice cultures.

Cryopreservation of an embryogenic suspension culture of Picea sitchensis and subsequent plant regeneration

Embryogenic suspension cultures of sitka spruce (Picea sitchensis) were successfully cryopreserved. The best method for cyropreservation as demonstrated by regrowth tests was: 1) preculture in medium containing 0.4 M sorbitol and pretreatment with 5% (v/v) DMSO (dimethylsulfoxide), 2) freezing the cultures at a cooling rate of 0.5°Cmin‐1 to —40°C followed by transfer to LN (liquid nitrogen). After thawing in a waterbath at 45°C the growth rates of cell cultures cryopreserved by this method were equal to growth rates of untreated unfrozen cultures. The cryopreserved cultures gave rise to mature somatic embryos which developed cotyledons, roots and shoots 3–4 months after thawing.

Cryopreservation of asparagus ( Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by vitrification

Embryogenic cells of asparagus ( Asparagus officinalis L.) were successfully cryopreserved by vitrification and subsequently regenerated plants. The cells were cryoprotected with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C for 10 min and then transferred to 1.8-ml plastic cryotubes. The cryoprotected cells were dehydrated with a new vitrification solution (designated PVS3) at 0°C for 20 min prior to a plunge into LN 2. PVS3 contains 50% (w/v) glycerol and 50% (w/v) sucrose in water. The vitrified cells in LN 2 were rapidly warmed in a water bath at 40°C and then expelled into LS medium supplemented with 1.2 M sucrose. The cell survival evaluated by fluoresceine diacetate and phenosafranin amounted to 80–90% of untreated, unfrozen controls. Revived cells resumed growth within 3 days after plating and regenerated plantlets via embryogenesis. Plant regeneration efficiency was nearly the same as that of the untreated, unfrozen control. Even an 80% concentration of PVS3 diluted with water produced 80% survival.

Catalase isozymes are useful markers of differentiation in maize tissue cultures

To examine the temporal pattern of catalase expression during callus induction, cultures obtained from immature embryos and leaves of several maize inbred lines at several times after explanting were evaluated. The results indicate that irrespective of the primary explant used, CAT-1 is the predominant isozyme in established callus. Catalase expression during the egeneration phase in the absence of auxin was assayed in the cultures. CAT-2 was found to be the isoform specific to this developmental time. The results indicate that different catalase isozymes are expressed at tissue culture level and callus initiation and subsequent plant regeneration can be easily monitored by means of catalase isozymes.

Twenty Years of Ecosystem Reorganization Following Experimental Deforestation and Regrowth Suppression

Watershed 2 of the Hubbard Brook Experimental Forest was experimentally deforested in the winter of 1965—1966, and subsequent plant regeneration was suppressed by herbicide application until the 1969 growing season. Changes in species structure, plant abundance, aboveground primary productivity and biomass, and aboveground nutrient pools were recorded on 70 permanent plots distributed across the watershed during 1, 2, 3, 5, 11, and 20 yr of subsequent succession. Species richness increased rapidly over time but equitability and the Shannon—Wiener diversity index declined with succession. Plant densities of herb, shrub, sapling, and tree strata increased, then decreased, in successive waves as the vegetation increased in height. Basal area (stems >5.1 cm dbh) initially increased exponentially, then linearly after the 5th yr to attain a total of 18.7 m2/ha by the 20th yr. Primary productivity also increased exponentially at first, but then increased linearly through the 20th yr at which time annual aboveground primary productivity was 20 Mg/ha. Aboveground biomass increased linearly after the 5th yr until by the 20th yr it was 52 Mg/ha, 38% of the reference forest biomass. Aboveground nutrient pools of some elements accumulated faster than biomass by the 20th yr when calculated as percentages of the 55—yr—old reference forest pools and biomass. These included: potassium–52%, phosphorus–44%, magnesium–42%. Other accumulated more slowly than biomass: sulfur–32%, and nitrogen and calcium each–29%. While recovery rate on Watershed 2 was delayed in the first decade of regrowth, and composition was somewhat altered because of the 3—yr suppression, this forest nevertheless displayed a vigorous capacity for regeneration after suppression ceased.

Multiple patterns of mtDNA reorganization in plants regenerated from different in vitro cultured explants of a single wheat variety

We have previously shown that tissue cultures derived from various explants of the wheat variety Chinese Spring exhibit organ/tissue-specific changes in the organization of their mitochondrial genome. The aim of this work was to study the influence of passage out of in-vitro culture, and subsequent plant regeneration, on the in vitro "induced" reorganization of this genome. In all cases but one, subgenomic configurations present in both the donor parent and the tissue culture were evident, in corresponding regenerated plants. The presence, in regenerated plants, of subgenomic configurations found in tissue culture but undetectable in the donor parent appeared to be both timeand organ/tissue-dependent. Moreover, when present, these novel organizations were not systematically found in all regenerated plants. Finally, novel subgenomic configurations were specifically detected after passage out of in-vitro culture. As these results were obtained from a single plant variety, they clearly confirm the extreme plasticity of mitochondrial genome structure in response to in-vitro culture.

Somatic embryogenesis and plant regeneration in Anthurium andraeanum hybrids

A method for the production of somatic embryos and subsequent plant regeneration for Anthurium andraeanum Linden ex André (Monocotyledonae) hybrids is described. Whole leaf blade explants, derived from plantlets grown in vitro, formed translucent embryogénic calli at their basal ends within one month of culture in the dark. Secondary somatic embryos formed frequently and without an intervening callus on surfaces of primary embryos. Embryogenesis was induced with three genotypes using a modified half-strength Murashige and Skoog (MS) medium supplemented with 1.0 to 4.0 mg l(-1) 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.33 to 1.0 mg l(-1) kinetin. A combination of 2% sucrose with 1% glucose in the medium favored embryogenesis over 3% sucrose alone. Whole leaf blades on medium solidified with 0.18% Gelrite produced more somatic embryos than leaves on medium with 0.7% Bacto-agar. Within two to three months after culture initiation, embryos were transferred to modified MS medium containing 0.2 mg l(-1) 6-benzyladenine (BA) and 2% sucrose and placed in the light for conversion into plantlets. Rooted plantlets were recovered and transferred into pots with tree fern fiber medium and grown in the greenhouse.

Induction of division and differentiation of somatic embryos in the leaf epidermis of Gaillardia picta

Somatic embroys and subsequent plant regeneration were obtained from isolated leaf epidermis of Gaillardia picta. Abaxial and adaxial epidermal peels (monolayer) from 45 days old aseptic seedlings were isolated and segments measuring 5 mm x 3 mm were cultured on B5 basal medium supplemented with various growth regulators such as naphthaleneacetic acid or indolebutyric acid and benzylaminopurine or kinetin. Within 12 h of culture the epidermal cells showed receding of cytoplasm from the walls. After 48 h of incubation 3 or 4 localized zones, each consisting of 3-8 cells that accumulated cytoplasm and stained densely, were observed. Mitotic divisions occurred in these zones on day 3 of culture and localized masses of callus were observed in 95% of the cultures after 10 days. In another 5 days, the callus differentiated somatic embryos or roots, depending on the growth regulators and their concentration in the medium.

Anther callus induction and green plant regeneration at high frequencies from an interspecific rice hybrid Oryza sativa Linn. x O. rufipogon Griff

Callus induction and green plant regeneration at high frequencies from an interspefic hybrid, Oryza sativa L. x O. rufipogon Griff. has been achieved by simply coordinating the growth regulators in the induction medium. The study was conducted with two different basal media (Potato-2 and N6) and seven different combinations of growth regulators 2,4-D, NAA and kinetin. Synergistic effects of the two auxins in enhanced anther response to callus induction and subsequent green plant regeneration were observed in both media. The highest frequency of callus induction was obtained on Potato-2 medium supplemented with 1 mg/12,4-D, 2 mg/l NAA and 1 mg/l kinetin. The same combination of growth regulators which yielded higher frequencies of callus induction also induced higher mean number of calli per anther. Although the calli formed on N6 medium showed high regenerability, there was a concomitant increase in the number of albinos among the regenerants. The auxins in the induction media had considerable influence on the regeneration capacity of the calli. The regeneration frequencies were higher from calli formed in the presence of both auxins in the induction media. The levels of growth regulator combinations seem to influence the green plant regeneration especially for calli induced on Potato-2 medium. Among the pollen grain derived plants the majority were either haploids or double haploids and very few were chromosomal variants.

Varietal Differences in the Ability of Callus Formation and Plant Regeneration from Mature Embryo in Barley (Hordeum vulgare L.).

In vitro callus formation by mature embryo explants, and subsequent plant regeneration were investigated using 203 Japanese and introduced varieties of barley (Hordeum vulgare L.). These included 176 six-rowed and 27 two-rowed genotypes. Mature embryos detached from seeds were inoculated on the modified N6 medium containing 2 mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D). Callus was produced in 193 varieties, although most of the calli were soft, watery, translucent and poor in proliferation. Plants could be regenerated from secondary callus especially with some two-rowed genotypes. The growth of secondary callus was promoted by increasing 2, 4-D concentration to 5 mg/l. Plant regeneration was observed in 14 varieties including 6 six-rowed and 8 two-rowed ones. Some of the varieties used, especially the two-rowed ones: may be useful for developing the mature embryo culture system in barley.

Cryopreservation in liquid nitrogen of cultured navel organe (Citrus sinensis Osb.) nucellar cells and subsequent plant regeneration

Suspension cultured cells of nucellar callus of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved. The nucellar cells were cryoprotected in Murashige-Tucker basal medium supplemented with 5% DMSO+1.2 M sucrose in an ice bath for 1 h, and then were frozen in this solution at a cooling rate of 0.5°C/min to −40°C prior to immersion in LN2. After rapid thawing in a +40°C water bath, regrowth was achieved by transferring the treated cells, without washing, onto filter paper discs over nutrient media solidified with agar. The viability after thawing, as evaluated by FDA and phenosafranine double staining, was about 70% of controls. The revived cells resumed growth within 3 days and produced cotyledonary embryos that developed into plants within 2 to 6 months of culture. Plants regenerated from cryopreserved cells were morphologically uniform and had the characteristics typical of navel orange.

Long-term callus cultures of diploid Barley (Hordeum vulgare). I. Auxin effects on culture initiation and maintenance

Use of 2,4-D was superior to NAA or IAA for embryogenic callus initiation or maintenance in barley cultivar Bruce. A concentration of at least 2.0 mg/l 2,4-D was desirable for culture initiation. The developmental size of the embryo was more important than embryo age for obtaining embryogenic calli. Even brief exposures (20–40 days) of calli to concentrations of higher than 5.0 mg/l 2,4-D or 10.0 mg/l NAA resulted in inhibition of subsequent plant regeneration and therefore, concentrations above these could not be used for maintenance cultures. In the long-term maintenance cultures, the best production of embryogenic calli was with 0.1 mg/l and 1.0 mg/l 2,4-D.

In vitro plant regeneration and ethylmethanesulphonate (EMS) uptake in somatic embryos of date palm (Phoenix dactylifera L.)

The effect of the auxins dicamba (3,6-dichloro-2-methoxybenzoic acid) and picloram (4-amino-3,5,6-trichloropicolinic acid) on callus growth and embryogenesis in Phoenix dactylifera L. was investigated. Maximum callus fresh weight was obtained in nutrient medium enriched with 200 µm picloram. Somatic embryogenesis and subsequent plant regeneration was achieved following transfer of such calli to hormone-free medium. Germination of the somatic embryos was influenced by treatment with the chemical mutagen ethylmethanesulphonate (EMS). Uptake of the labelled mutagen ([14C]EMS) by the somatic embryos increased with increased incubation time. Presence of dimethyl sulphoxide (DMSO) as a carrier agent during mutagenic treatment was necessary for efficient mutagen uptake.

High frequency somatic embryogenesis and plant regeneration from immature inflorescence cultures of two Indian cultivars of sorghum (Sorghum bicolor L. Moench)

Somatic embryogenesis and subsequent plant regeneration were obtained from immature inflorescence segments of two cultivars ofSorghum bicolor L. Moench. Callus was induced on Murashige and Skoog’s medium supplemented with 2,4-dichlorophenoxyacetic acid (2 mg/l) and zeatin (0.1 mg/l). Transfer of calli to Murashige and Skoog medium alone or with reduced level of auxins with or without cytokinins resulted in the production of somatic embryos and plantlets. The combination of a cytokinin with Triiodobenzoic acid promoted the highest number of somatic embryoids. The best response was obtained from inflorescences 10–25 mm in length. Regenerated plants were transferred to field for further evaluation.