Selection and characterization of a new switchgrass (Panicum virgatum L.) line with high somatic embryogenic capacity for genetic transformation

Switchgrass (Panicum virgatum L.) is used horticulturally as an ornamental and agronomically as an animal feedstock and a putative bio-energy crop. Genetic transformation, using somatic embryogenic (SE) callus derived from mature seeds, is one strategy for improving switchgrass traits. A superior switchgrass line, HR8, was developed in this study using recurrent tissue culture selection from cv. Alamo. Eighty two percent of HR8 seeds germinated after harvest comparing to 26.8% for unselected ‘Alamo’. HR8 seeds that germinated produced 84.9% SE callus. HR8 seeds had higher endogenous abscisic acid (ABA) contents and responded differently to exogenous additions of ABA in culture. Endophytes were isolated from switchgrass seeds and callus. HR8 callus had less endophytic contamination than that of ‘Alamo’ callus. HR8 SE calli were genetically transformable using Agrobacterium. Therefore, HR8 is a superior line for generating SE callus and Agrobacterium-mediated transformation.     

Plantlet formation in callus tissues of Anthurium andraeanum Lind.

A tissue culture method is described for the vegetative propagation of Anthurium andraeanum Lind. through callus tissue differentiation. The callus tissue was induced from excised embryos and young parts of adult plants, cultivated on a modified Murashige and Skoog's medium supplemented with the cytokinin PBA. Optimum growth of callus tissue was obtained at 25°C in darkness, but the rate of growth was highly variable amongst callus clones obtained from different genotypes. Callus tissue could be subcultured and induced to form adventitious sprouts, especially in light. These sprouts regenerated roots on the basic culture medium. The first Anthurium clones have been transferred to pots and are now growing vigorously in the greenhouse.     

Callus induction and culture of Rosa

Leaf and stem explants of Rosa manetti Hort. and R. hybrida L. ‘Tropicana’ were placed on Murashige and Skoog (MS), Schenk and Hildebrandt (SH) and Gamborg and Wetter (1-B5C) media, containing growth regulators, casein hydrolysate (CH) or coconut milk (CM), to determine the optimum conditions for callus initiation and maintenance. The explants were cultured either in dark or in light (2 Klux 16 h/day) at 26±2°C. Friable, fast growing callus was evident after 3 weeks for both rose species on MS + 2.0 mg/l 2,4-D, 0.25 mg/l kinetin (K) and 2.0 g/l CH as well as on SH + 0.5 mg/l 2,4-D, 2.0 mg/l p-CPA and 0.1 mg/l K. Callus initiation occurred sooner on SH than on MS. However, during sub-culture of callus arising on SH, rapid oxidation often occurred, resulting in severe browning of the callus, which made it unsatisfactory for further experimental use. Callus initiation occurred faster in dark than in light, but deteriorated when continuously sub-cultured in the dark regardless of media. Although ‘Tropicana’ initiated callus sooner than R. manetti, very fast growing callus of the latter was obtained when leaf callus was transferred to MS + 2.0 mg/l NAA and 0.5 mg/l BA. Unsuccessful attempts to regenerate callus and induce adventitious shoots on the 2 explants are discussed.     

Histology of adventitious shoot and root formation on leaf-petiole cuttings of Begonia x hiemalis Fotsch ‘Aphrodite Peach’

Epidermal and subepidermal cells formed a callus at the basal portion of the petiole of Rieger Begonia cv. ‘Aphrodite Peach’ leaf-petiole cuttings. Roots arose from cells of the internal portion of the callus and also from parenchymal cells of the petiole. Shoots formed from cells on the surface of the enlarging callus. Inasmuch as many cells had divided in the callus before an organized shoot appeared, it was virtually impossible to determine from histological observations whether the cells of the new shoot were derived from a single epidermal cell of the petiole or from multiple epidermal cells. These histological observations on adventitious shoot formation are discussed relative to mutation breeding employing leaf-petiole cuttings of B. x hiemalis Fotsch.     

Regeneration of different Cyclamen species via somatic embryogenesis from callus,suspension cultures and protoplasts

The present study is the first report of the establishment of embryogenic callus cultures from seedling tissue, the regeneration of plants via somatic embryogenesis and the development of a regeneration system from protoplast to plant, using three wild species of Cyclamen, Cyclamen graecum Link, Cyclamen mirabile Hildebrand, Cyclamen trochopteranthum Schwarz (syn. Cyclamen alpinum hort. Dammann ex Sprenger). The ability to form embryogenic callus and to regenerate via somatic embryogenesis was strongly genotype-dependent for each species. From 0.5 g callus, up to 1461 somatic embryos were formed in the case of C. mirabile. Culture media with different concentrations of plant growth regulators, CaCl₂ and activated charcoal significantly influenced embryo formation in this species. Up to 1.4 × 10⁶ protoplasts were isolated from 1 g of C. graecum cell suspension. Diverse growth responses of the protoplasts in two embedding agents, agarose and alginate, were observed for the different Cyclamen species. These specific growth characteristics could be used as a selection marker for future fusion experiments. From both protoplast culture systems, somatic embryos were regenerated, grown to plantlets and acclimatised to greenhouse conditions.     

Increased haploid production in Saintpaulia ionantha by anther culture

Anthers of Saintpaulia ionantha containing late-uninucleate stage pollen produced callus from the anther interior after 3–4 weeks culture on Murashige and Skoog medium supplemented with 1 mg l^?1 naphthylene acetic acid (NAA) and 0.5 mg l^?1 1,6-benzylaminopurine (BAP). Shoot regeneration occurred rapidly and up to 200 shoots could be recovered from callus derived from a single anther within 10 weeks. Examination of roottip mitoses from transplanted, established plants demonstrated that, with few exceptions, the plants were haploid, thus indicating that the callus was pollen derived. Exposure of buds to low temperature prior to anther excision was inhibitory to callus production. Shoot regeneration was studied by scanning-electron microscopy.     

In vitro propagation of three endangered cactus species

We tested the feasibility of in vitro culture techniques for the propagation of the three endangered cacti species Escobaria minima (Baird) D. Hunt, Mammillaria pectinifera (Ruempler) F.A.C. Weber and Pelecyphora aselliformis Ehrenberg. Twenty-five MS-based proliferation media were tested in preliminary experiments, with different combinations of the auxin NAA and either of the cytokinins BA, kinetin or TDZ. TDZ induced a good proliferation rate, albeit associated with abundant callus formation and hyperhydricity of axillary shoots. A high multiplication rate combined with good quality proliferated shoots and little or no callus induction was observed on media containing BA for E. minima and M. pectinifera and on a medium containing kinetin for P. aselliformis. These results were also confirmed in subsequent experiments in which different explants (shoot tips, bases and longitudinal sections) were used. Micropropagated plantlets were successfully restored to the field, where they reached the flowering stage. Plantlet regeneration of M. pectinifera and P. aselliformis from callus induced on media containing TDZ, but not 2,4-D, was also achieved.     

Anther culture in rice proportionally rescues microspores according to gametophytic gene effect and enhances genetic study of hybrid sterility

Background

To investigate plant hybrid sterility, we studied interspecific hybrids of two cultivated rice species, Asian rice (Oryza sativa) and African rice (O. glaberrima). Male gametes of these hybrids display complete sterility owing to a dozen of hybrid sterility loci, termed HS loci, but this complicated genetic system remains poorly understood.

Results

Microspores from these interspecific hybrids form sterile pollen but are viable at the immature stage. Application of the anther culture (AC) method caused these immature microspores to induce callus. The segregation distortion of 11 among 13 known HS loci was assessed in the callus population. Using many individual calli, fine mapping of the HS loci was attempted based on heterozygotes produced from chromosome segment substitution lines (CSSLs). Transmission ratio distortion (TRD) from microspores was detected at 6 of 11 HS loci in the callus population. The fine mapping of S₁ and S₁₉ loci using CSSLs revealed precise distances of markers from the positions of HS loci exhibiting excessive TRD.

Conclusions

We demonstrated that AC to generate callus populations derived from immature microspores is a useful methodology for genetic study. The callus population facilitated detection of TRD at multiple HS loci and dramatically shortened the process for mapping hybrid sterility genes.

     

De novo differentiation of shoot buds from leaf-callus of Dianthus caryophyllus L. and control of hyperhydricity

A method is described for producing de novo shoots from leaf derived callus of carnation (Dianthus caryophyllus L.). Plants were regenerated in four steps, viz. callus induction, shoot regeneration, removal of hyperhydricity from regenerated shoots and root development. Callus induction medium contained 2,4-D and BAP. Shoot buds were formed when the callus was further subcultured on 2,4-D- and BAP-containing medium, or MS medium without any growth regulators. The shoots so formed were hyperhydric, bushy in appearance with reduced stem length and watery leaves. The normal conformation of shoots was restored by culturing the hyperhydric shoots onto medium supplemented with GA₃ and bactopeptone. The recovered shoots were rooted on MS medium added with NAA (1 mg/l) or IBA (2 mg/l). Regenerated plants with well-developed root and shoot systems were successfully transferred to field conditions after initial acclimation.     

Plantlet regeneration and stability from callus cultures of Freesia × hybrida Bailey cultivar ‘Royal’

True-to-type plantlets of Freesia × hybrida Bailey cultivar ‘Royal’ were generated from callus after 27 months of sub-culture in liquid medium. Callus was initiated from young flower pedicels cultured on semi-solid Linsmaier—Skoog (LS) medium supplemented with 5 mg/l of 2,4-D and 0.5 mg/l kinetin, and transferred to the same medium in liquid form without hormones and thereafter sub-cultured every 7–10 days. Liquid cultures with 2.4–4.3 g of callus per 25 ml medium produced largest increases in callus fresh weight. Callus generated the most shoots when cultured on LS medium supplemented with 5 mg/l of kinetin and incubated in the light, while fewer plantlets were produced when no growth regulator or GA₃ or PBA were used. Callus cultures incubated in continuous darkness did not form shoots.     

Screening of vincristine yield in ex vitro and in vitro somatic embryos derived plantlets of Catharanthus roseus L. (G) Don.

Catharanthus roseus L. (G.) Don. (Apocynaceae) is an important dicotyledonous medicinal plant as it is the sole source of vincristine and vinblastine that are used against a variety of cancers. Quantitative estimation of vincristine was carried out using high performance liquid chromatography (HPLC) in various in vitro grown tissues; calluses (embryogenic and non-embryogenic callus), embryogenic stages (proliferated, matured and germinated embryos), somatic embryo derived plantlets (leaf, root and whole plant) and leaves of ex vitro developed plantlets. The yield in those in vitro and ex vitro-developed tissues was monitored for 30 weeks. Except at an early lag period, vincristine production was detected up to 20–25 weeks old plant samples. Vincristine content was very high in leaf callus and germinated embryos. Leaves of in vitro raised plants showed higher amount of vincristine when compared to ex vitro-developed leaves of similar age. Vincristine production was tissue specific and age dependent that was discussed in detail in this present communication.     

In vitro culture of cotyledon tissue of Castanea sativa Mill

Establishment of callus tissue culture from cotyledon fragments of Castanea sativa Mill. is described. The explants were grown on various combinations of auxins (IAA, IBA, NAA, 2,4-D) and cytokinins (K, BA) to induce callus and regeneration of organs. The best growth occurred on basal medium with 2,4-D (1 and 10 mg/l) plus both K or BA (0.5 mg/l). Root regeneration was achieved with both IBA and NAA (10 mg/l) supplemented with K or BA.     

Using a gestalt to measure the quality of in vitro responses

Overall “quality” of in vitro responses can sometimes be difficult to assess using measured response variables (e.g., shoot number and height, and callus fresh weight). Gestalt Theory is the idea that the whole is perceived to be greater than the sum of the individual parts. To determine if a gestalt assessment could be used to assess quality of in vitro responses two plant tissue culture systems were examined—Brugmansia x candida shoot multiplication and sweet orange nonembryogenic callus growth. The gestalt assessment of each system was compared to measured responses—shoot number, leaf length and width for Brugmansia x candida, and fresh weight accumulation for citrus. The gestalt analysis modeled as precisely as the measured response variables for both in vitro systems while satisfying the statistical assumptions necessary for a valid analysis. We concluded that the gestalt response is a valid response as it was indistinguishable, in terms of statistical quality, from the measured responses.     

Influence of yeast extract and casein hydrolysate on callus multiplication and somatic embryogenesis of date palm (Phoenix dactylifera L.)

Complex organic additives are known to improve growth and differentiation of in vitro plant cultures. The present investigation was conducted to determine the effect of various concentrations of yeast extract (YE) and casein hydrolysate (CH) on callus growth and somatic embryogenesis in date palm cultivar Nabout Saif. Callus induced from shoot tip explants was grown on callus multiplication medium supplemented with either YE or CH at 0.0, 0.1, 0.25, 0.5, and 1 g l⁻¹. To induce somatic embryogenesis, callus was transferred to a hormone-free medium containing the corresponding concentration of additives. The results have shown that callus weight and the number of somatic embryos were directly proportional to increases in the concentration of organic additives tested. Callus growth was best achieved when 1 g l⁻¹ of either YE or CH was added to the culture medium. At this concentration of YE, callus growth was double that of the control medium. On CH-containing media growth was 2.3 times that of the control. This indicates that CH is more effective in enhancing callus growth. However, YE was more effective in enhancing somatic embryogenesis. The data show that the best somatic embryo formation was obtained on either 1 g l⁻¹ YE or 0.5 g l⁻¹ CH which produced 45 and 30 embryos per culture, respectively, as compared to 20 embryos produced in the control treatment. Resultant somatic embryos successfully rooted and regenerated plantlets which exhibited normal growth in the greenhouse. Enhanced plant regeneration, an essential criterion for commercial micropropagation, was achieved.     

Physiological response of grapevine cultivars and a rootstock to infection with Phaeoacremonium and Phaeomoniella isolates: an in vitro approach using plants and calluses

In vitro plants and callus culture of two Vitis vinifera cultivars (cv. Baga and Maria Gomes) and one rootstock (R3309, i.e. Vitis riparia var tomentosa × Vitis rupestris) were inoculated with conidia of Phaeoacremonium angustius and Phaeomoniella chlamydospora. Response to infection was determined in plants grown in vitro by assaying growth rates, malondialdehyde (MDA) production (lipid peroxidation) and chlorophyll content and fluorescence. Growth rate and malondialdehyde production were also used to determine resistance of calluses to infection. Infection reduced growth and increased MDA in infected plants and calluses, and reduced chlorophyll content and fluorescence in infected leaves. Symptoms were more evident in plants infected with P. angustius, showing that this species is more virulent to plants and calluses than Ph. chlamydospora. Differences in virulence among strains of Ph. chlamydospora were also found, as 1AS and CAP053 were more virulent (induced more severe decreases of growth and chlorophyll fluorescence, together with higher MDA production in both cultivars) then CAP080. Growth of rootstock plants and calluses was less affected by infection than growth of other cultivars. Contrarily to Baga and Maria Gomes, chlorophyll content and fluorescence of rootstock plants were only affected by P. angustius. Also Baga plants and calluses were more resistant than those of Maria Gomes. These data show different degrees of resistance among genotypes. Reduction of callus production by infection supports the idea that fungus infection may reduce cicatrisation by inhibiting callus formation during grafting or wounding; and therefore, contribute to the entrance of opportunist pathogens. Implications of using in vitro cultures to assay host/pathogen relationship and virulence/resistance degrees among the different genotypes of fungus and grapevines are discussed.     

In vitro stable regeneration of onion and garlic from suspension culture and chromosomal instability in solid callus culture

An efficient regeneration system from bulb-derived callus tissues in suspension of onion (Allium cepa L.) and garlic (A. sativum L.) was established. Callus culture was induced in Gelrite^®-solidified Murashige and Skoog's (MS) modified basal medium with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.93 μM kinetin supplements. After 4 weeks of induction, the callus tissue was partly transferred to liquid MS media containing different levels of α-naphthaleneacetic acid (NAA) and kinetin for plant regeneration and the rest was maintained in the same medium for chromosome analysis and nuclear DNA quantification following in situ microspectrophotometry. The cultures in suspension, maintained in agitated condition for 8 weeks, showed a high frequency of rapidly regenerated plants after transferring to Gelrite^®-solidified one half strength of MS basal medium. Chromosome analysis of the regenerated plants, transferred to the field with 90% survival rate, revealed stable chromosome number (2n = 16) in both species. On the other hand, callus tissues maintained in solid induction medium for long period showed abnormality in chromosome behavior leading to the formation of both hypo- and hyper-diploid cells along with the diploid cells. The frequency of aneuploid cells (2.2–48.9%) increased with callus age in both species with high and statistically significant number of hyperdiploid cells. The role of endoreduplication as well as non-disjunction of chromosomes resulting in instability in chromosome number has been suggested. This was also supported by the nuclear DNA value in successive passages with statistically significant increase.     

Morphogenetic responses of Salpiglossis sinuata L. leaf explants and callus

Leaf explants of Salpiglossis sinuata L. were cultured on MS medium containing the auxins 2,4-D, IAA, NAA, or the cytokinins 6-BAP, 2ip, or K singly or NAA + 6-BAP combinations (0.01 to 10.0 mg/l) to determine their morphogenetic responses. IAA and NAA induced callus or roots at all levels except IAA at 0.01 mg/l. Callus varying in friability was obtained on MS + 2,4-D at various concentrations. Friable, uniform dividing callus was obtained on UM medium. Optimum adventitious shoot formation occurred on leaf sections and from callus cultures placed on MS + 2ip. Rooting-difficulties were encountered, but 75% rooting efficiency was obtained on shoots cultured in MS + 0.001 mg/l 2,4-D. Rooted plants were readily transferred to the greenhouse, and flowered.     

Flavonol glycosides of different species and hybrids from the Prunus section eucerasus and the growth-promoting activity of quercetin derivatives

The total content of flavonol glycosides was lowest in P. avium L., medium in P. cerasus L. and highest in P. fruticosa Pall. The hybrids between P. fruticosa × P. avium and P. cerasus × P. fruticosa showed an intermediate pattern. Quercetin glucoside and quercetin rhamnoglucoside rutin) occurred regularly in all trees investigated. Some flavonol glycosides could not be identified exactly.Young shoot segments of P. avium (F12/1) were grown for 4 weeks in vitro in continuous darkness or at a day length of 16 hours. The basal medium was supplemented with several flavonoids. Rutin and dihydroquercetin stimulated the callus growth. The tissue was unresponsive to quercetin and kaempferol. Among the factors contributing to the callus production were the light regime as well as the growth factors of the medium.     

Callus induction and plant regeneration from embryonic axes of Kosteletzkya virginica

Kosteletzkya virginica, a perennial dicot halophytic species of the Malvaceae, is native to American salt marsh. It was introduced into China as a potential species to improve coastal wetlands and to develop ecologically sound saline agriculture. K. virginica adapts excellently to the tidal-flat habitats in China's east coast, with multiple eco-benefits; in particular, its seed oil could be used to produce biodiesel. The purpose of this study was thus to develop a standardized protocol to induce a high frequency of callus and subsequent plantlet regeneration system for a K. virginica breeding program with the final objective of applying transgenic techniques to improve seed oil yield. The embryonic axes of K. virginica were used as explants for callus induction, shoot induction from the callus and then adventitious root induction from the shoots on nine culture media with different hormone combinations. The best results were achieved on the following media: (1) 93.94% callus induction on MS medium supplemented with 1.0 mg L⁻¹ indole-3-acetic acid (IAA), 0.3 mg L⁻¹ kinetin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (2) 65.83% shoot induction on 1/2MS medium supplemented with 0.1 mg L⁻¹ IAA, 0.5 mg L⁻¹ zeatin, 30 g L⁻¹ sucrose and 8 g L⁻¹ agar; (3) 96.67% rooting on MS medium containing 30 g L⁻¹ sucrose and 8 g L⁻¹ agar. The survival rate of plantlets by organogenic regeneration was 85% after being transplanted into potting soil in flowerpots and placed in the greenhouse. This experiment indicates that we established successful callus induction and plant regeneration protocols for K. virginica.