Somatic Hybrids of Sunflower (Helianthus annuus L.) Identified at the Callus Stage by Isoenzyme Analysis

Dark grown hypocotyl protoplasts from Helianthus annuus L. cvs. Cerflor and Euroflor were electrically fused to produce somatic hybrids. Following fusion, the protoplasts were cultivated in agarose droplets for four weeks. Macroscopic visible calli (0.1-0.3 mm) were transferred onto solid medium and calli reaching a size of 3 mm were collected. Their isoenzyme patterns were analysed based on two different isoenzymes that allow discrimination between the two cultivars used for fusion. From the examined calli, about 26 % showed an isoenzyme pattern of putative binary heterokaryocytes. The isoenzyme pattern of the non-fused control revealed less than 5% of possibly chimeric colonies.     

2,4-Dichlorophenoxyacetic acid affects mode and frequency of regeneration from hypocotyl protoplasts ofBrassica oleracea

Summary The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on the regeneration from hypocotyl protoplasts ofBrassica oleracea was studied by varying the 2,4-D concentration in the protoplast culture medium, 8 p, and the callus proliferation medium, K3. When hypocotyl protoplasts of the inbred line BL12 were cultured in the complete absence of 2,4-D, they divided and produced embryogenic calli. Moreover, these calli generated somatic embryos which were easily recognized by red cotyledons due to the presence of anthocyanin. When 2,4-D was present either in 8p medium or K3 medium the formation of somatic embryos was reduced. On the other hand, the number of shoot-forming calli increased considerably. We therefore conclude that 2,4-D directs the mode of regeneration by suppressing somatic embryogenesis in favour of shoot regeneration. Secondly, 2,4-D increases the regeneration efficiency. Furthermore, the callus proliferation phase on K3 medium is most important with respect to the determination of either somatic embryogenesis or shoot regeneration.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole acetic acid - NAA naphthalene acetic acid - PE plating efficiency     

Effects of brassinosteroid on cotton regeneration via somatic embryogenesis

Brassinolide (BR), which is the most biologically active brassinosteroid, was used to examine the potential effect of hormone on cotton somatic embryogenesis. Ten-day-old cotton (Gossypium hirsutum L., cv. Cooker) seedlings were used for explant source and hypocotyls were removed and cultured on MS basal medium with B₅ vitamins supplemented with 1 mg/L 6-benzylaminopurine + 0.5 mg/L kinetin for callus induction. After one month proliferating calli pieces were collected and cultured on MS basal medium containing various concentrations of BR (0.1, 0.5, 1.0 μM) with their controls. BR treatments were negatively effective on the fresh weight of calli when compared to control. Differential somatic embryogenesis maturation rates due to BR treatment were observed. Somatic embryogenesis was stimulated especially for transition to cotyledonary phase at 0.5 mg/L BR. Histological preparations from embryogenic calli and somatic embryos at different stages of development revealed the spontaneous polyploidisation during early somatic embryogenesis on BR-treated calli. Present results suggest that BR negatively effected calli growth, however, had a stimulating role in maturation of somatic embryos.     

Somatic embryogenesis,organogenesis and callus growth kinetics of flax

The effects of plant growth regulators (PGR) on calli induction, morphogenesis and somatic embryogenesis of flax were studied. The organogenic and callus formation capacity were assessed for different types of source explants. Root and shoot explants were equally good material for calli production but the former produced calli without shoot regeneration capacity. Under the experimental conditions tested, 2,4-dichlorophenoxyacetic acid (2,4-D) + zeatin was the most efficient PGR combination on calli induction and biomass production. The calli were green but with no rhizogenic capacity. In contrast, and at similar concentrations, indole-3-butyric acid (IBA) + kinetin induced white or pale green friable calli with a good root regeneration capacity (60%). A factorial experiment with different combinations of 2,4-D + zeatin + gibberellic acid (GA₃) levels revealed that the direction of explant differentiation was determined by specific PGR interactions and concentrations. The results from these experiments revealed that the morphogenetic pathway (shoot versus root differentiation) can be manipulated on flax explants by raising the 2,4-D level from 0.05 to 3.2 mg l^?1 in the induction medium. The induction and development of somatic embryos from flax explants was possible in a range of 2,4-D + zeatin concentrations surrounding 0.4 mg l^?1 2,4-D and 1.6 mg l^?1 zeatin, the most efficient growth regulator combination.     

Somatic embryogenesis from leaf protoplasts of Rauvolfia vomitoria shoot cultures

Rauvolfia vomitoria mesophyll protoplasts have been isolated from axenic shoot cultures and cultured (10⁵-10⁶ protoplasts per ml) in Murashige and Tucker liquid medium containing growth regulators. Within 6–8 weeks, a mixed population of calli and proembryos were obtained and transferred on solid media. Calli produced shoots; however, rooting did not occur. Somatic embryos achieved different patterns of development. In particular, whole plantlets have been obtained either directly through germination of primary embryos or via embryogenic calli.Abbreviations B5 Gamborg et al. (1968) medium - BA N⁶ (benzyl) adenine - 2,4-D 2,4 dichlorophenoxyacetic acid - MT Murashige and Tucker (1969) medium - NAA naphthalene acetic acid - Z zeatin - K kinetin     

Induction of somatic embryogenesis from female flower buds of elite <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were low, but increased in the presence of gibberellic acid (GA₃). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs). The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia, but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of somatic embryo origin.     

Plant regeneration from callus and protoplast cultures of Helianthus giganteus L.

Summary Methods of plant regeneration from callus and protoplasts of Helianthus giganteus L. are described. Embryogenic callus was obtained from leaf explants and plants were regenerated from these calli on MS media with different combinations of benzyladenine and naphtaleneacetic acid. Leaf protoplasts isolated from in vitro grown plants formed somatic embryos when cultured in agarose solidified droplets of V-KM medium containing benzyladenine and naphtaleneacetic acid. Embryos developed into plantlets on media with reduced auxin contents. Regenerated plants were successfully planted in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - MS Murashige and Skoog medium - NAA naphtaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls     

Protoplast isolation and culture for somatic hybridization of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> and <Emphasis Type="Italic">L</Emphasis>. <Emphasis Type="Italic">subcarnosus</Emphasis>

A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 10⁵ protoplasts g⁻¹ fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks) and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids. However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration.     

Stimulation of root and somatic embryo production in Euonymus europaeus L. by an inhibitor of polyamine biosynthesis

In vitro formation of roots and somatic embryos is obtained from cotyledon explants of a Spindle tree (Euonymus europaeus L.) cultured on two different media: a medium inducing callus formation and the production of roots, and a medium inducing callus formation, root and somatic embryo production. We studied the effects of -difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC) on root and somatic embryo production, growth and titers of putrescine in Euonymus explants and explant-derived calli. Early changes in putrescine levels were detected in both cultures before the visible emergence of roots or somatic embryos. DFMO rapidly inhibited putrescine accumulation and growth in non-embryogenic calli and highly stimulated rooting activity. DFMO partially inhibited putrescine accumulation in embryogenic calli. This inhibition had no effects on callus growth but significantly reduced the time of emergence of roots and highly stimulated somatic embryo production. The relationship among putrescine, putrescine metabolism, growth, root and somatic embryo formation is discussed.     

Intergeneric somatic hybrid plantlets between Dianthus barbatus and Gypsophila paniculata obtained by electrofusion

Hypocotyl-derived protoplasts of Dianthus barbatus that had been pretreated with iodoacetamide were fused electrically with cell suspension culture-derived protoplasts of Gypsophila paniculata that could divide to form callus but could not regenerate shoots under the culture conditions used in this study. Electrofusion-derived calli which produced shoots were selected as putative somatic hybrids, and plantlets were subsequently regenerated from 2 of these selected calli. These plantlets, which in vitro produced flowers precociously, were identified as intergeneric somatic hybrids by nuclear ribosomal DNA analysis. Normal plants have not been established up to the present.     

Optimization of direct somatic embryogenesis from mature zygotic embryos ofPanax ginseng C. A. Meyer

Culture conditions were optimized for somatic embryogenesis ofPanax ginseng. The highest frequency of embryo formation was obtained when tissues were excised from the middle region of the cotyledon segments of zygotic embryos. Only treatment with light could stimulate the formation of single-type somatic embryos, whereas multiple-type somatic embryos and calli were observed under dark conditions. The highest production of somatic embryos was found with an NH₄ ⁺:NO₃ ratio of 21:39. Among the tested media (MS, B5, and SH), maximum formation of somatic embryos was obtained when cotyledon expiants were cultured on an 1% agar MS medium supplemented with 5% sucrose. Regenerated ginseng plantlets were transferred to an autoclaved soil mixture in the greenhouse. These transformants showed no detectable variations in their morphology or growth characteristics compared with the donor plant.     

Somatic embryogenesis of <Emphasis Type="Italic">Byrsonima intermedia</Emphasis> A. Juss.: induction and maturation via indirect approach

Byrsonima, especially the species Byrsonima intermedia, is an endangered Brazilian plant that has been widely used as food and for its therapeutic characteristics. However, this species faces challenges with sexual propagation, and somatic embryogenesis has emerged as a viable alternative option for propagation. Therefore, this study aimed to establish a protocol for inducing somatic embryogenesis in B. intermedia. For the induction of callus from in vitro seedling leaves, different subcultures (three subcultures, 60 days each) and concentrations of different cytokinins (BAP, TDZ, Kin and ZEA) combined with varying NAA solutions were tested. Different concentrations of NAA were also analyzed in the induction of pro-embryogenic calli. For the induction of embryogenic calli and somatic embryos, the pro-embryogenic calli were subcultured on MS medium without adding growth regulators. The somatic embryos that originated were inoculated on a maturation medium containing different concentrations of gibberellic acid (GA₃). The formation of secondary embryos was also analyzed using different concentrations (0, 2.88, and 8.66 µM) of GA₃ and different types of lids (Conventional lid, Biossama® commercial lid and conventional lid with membranes). The results show that for the induction of somatic embryos, the use of kinetin with NAA presented the formation of somatic embryos in the second (4.76 µM CIN?+?0.54 µM NAA) and third (5.17 µM CIN?+?10.54 µM NAA) subcultures. The use of 28.87 µM GA₃ favored the formation of seedlings. The Biossama lid and 2.88 µM GA₃ showed higher formation of secondary embryos.     

Somatic hybridization between selected Lycopersicon and Solanum species

Summary Mesophyll protoplasts of an interspecific Lycopersicon esculentum Mill, (tomato) x Lycopersicon pennellii hybrid plant (EP) were fused with callus-derived protoplasts of Solanum lycopersicoides Dun. using a modified PEG/DMSO procedure. The EP plant was previously transformed by Agrobacterium tumefaciens which carried the NPTII and nopaline synthase genes. Protoplasts were plated at 10⁵/ml in modified KM medium and 16 days post-fusion 25 ug/ml kanamycin was added to the culture medium. During shoot regeneration, 212 morphologically similar putative somatic hybrids were delineated visually from kanamycin resistant EP's. Forty-eight shoots, randomly selected among the 212, were further verified as somatic hybrids by their leaf phosphoglucoisomerase heterodimer isozyme pattern. However, the resulting plants were virtually pollen sterile. In a second fusion, mesophyll protoplasts of Solanum melongena (eggplant) were fused with EP callus-derived protoplasts. Using the same fusion and culture procedure, only two dark green calli were visually selected among the pale green parental EP and verified as somatic cell hybrids by several isozyme patterns. These two calli have produced only leaf primordia in one and half years on regeneration medium.Abbreviations ABA abscisic acid - BAP 6 benzylaminopurine - 2,4-D 2,4 dichlorophenoxy acetic acid - DMSO dimethyl sulfoxide - GA₃ gibberellic acid - GOT glutamate oxaloacetate - IAA indoleacetic acid - IBA indolebutyric acid - IDH isocitrate dehydrogenase - MDH malate dehydrogenase - MES morpholinoethane-sulfonic acid - PEG polyethylene glycol - 6-PGDH 6 phosphogluconate dehydrogenase - PGI phosphoglucoisomerase     

Plant regeneration from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.)

A procedure is described to regenerate plants from embryogenic suspension-derived protoplasts of ginger (Zingiber officinale Rosc.). Somatic embryogenic calli were induced from ginger shoot tips on solid MS medium with half the concentration of NH₄NO₃ and supplemented with 1.0 mg l⁻¹ 2,4-Dichloroacetic acid (2, 4-D) and 0.2 mg l⁻¹ Kin. Rapid-growing and well-dispersed suspension cultures were established by subculturing the embryogenic calli in the same liquid medium. Protoplasts were isolated from embryogenic suspensions with an enzyme solution composed of 4.0 mg l⁻¹ cellulase, 1.0 mg l⁻¹ macerozyme, 0.1 mg l⁻¹ pectolyase, 11% mannitol, 0.5% CaCl₂ and 0.1% 2-(N-morpholino) ethane sulphonic acid (MES) for 12–14 h at 27°C with a yield of 6.27 × 10⁶ protoplasts g⁻¹ fresh weight. The protoplasts were cultured initially in liquid MS medium with 1.0 mg l⁻¹ 2, 4-D and 0.2 mg l⁻¹ Kin. Then the protoplast-derived calli (1.5 cm²) were transferred to a basal MS medium containing 0.2 mg l⁻¹ 2, 4-D, 5.0 mg l⁻¹ benzyladenine (BA), 3% sucrose and 0.7% agar. The white somatic embryos were transferred to MS medium lacking growth regulators for shoot development. Shoots developed into complete plantlets on a solid MS medium supplemented with 2.0 mg l⁻¹ BA and 0.6 mg l⁻¹ α-Naphthaleneacetic acid (NAA). In addition, the effects of AgNO₃, activated charcoal (AC) and ascorbic acid (AA) on browning of protoplast-derived calli are discussed.     

Protoplast fusion between Lycopersicon esculentum and L. peruvianum-complex: somatic embryogenesis, plant regeneration and morphology

Somatic hybrids were obtained by polyethylene glycol fusion of cotyledon protoplasts of Lycopersicon esculentum Mill. cv. Kyoryokutoko treated with iodoacetamide (IOA) and suspension-culture-derived protoplasts of L. peruvianum (PI270435) or L. chilense (PI128652). The hybrids were selected by a multiple-step selection procedure relying on the different colors of the fusion partners, IOA treatment of cotyledon protoplasts, and the use of a culture medium which only allowed cotyledon protoplasts to regenerate. The somatic embryos were derived from greenish calli that formed from the fusion mixtures, developed progressively through the globular, heart, and torpedo stages, and finally formed complete plantlets. The excised torpedo-stage embryos could be propagated on a modified medium. The morphology of the somatic hybrids were intermediate to their donor partners, and chromosome observations indicated that the hybrids were tetraploid, hexaploid, and aneuploid. Received: 24 July 1997 / Revision received: 4 November 1997 / Accepted: 2 December 1997     

Somatic embryogenesis and organogenesis in callus cultures of a tree legume — Albizia richardiana King

Hypocotyl explants of 1 and 10 mm lengths were excised from 12-day-old in vitro-grown seedlings of Albizia richardiana. The larger pieces, after 40 days of culture, developed shoots along with green calli on B₅ + BAP (10^–7–10^–5M), while the smaller segments produced only green calli on B₅+BAP (10^–7–10^–4M) medium. Some of the green calli turned morphogenic and started producing somatic embryos with the 2nd sub-culture and shoots from 7th sub-culture onwards. Calli retained the morphogenic potential even after repeated sub-culturing for over two years. The number of embryos in an embryogenic culture varied from 2 to 20 per callus mass of 5–6.5 cm³. Sucrose at the 2% level in MS medium was optimal for embryogenesis while 4% was optimal for shoot bud differentiation. Higher levels of sucrose (6–10%) caused browning of green calli and also inhibited differentiation into embryos and shoot buds. By selective sub-culturing of 0.1 cm³ pieces of embryogenic calli on MS+10^–5M BAP, 46% of the cultures produced somatic embryos. The latter germinated into plantlets on Knop's medium.Abbreviations BAP 6-benzylaminopurine - B₅ Gamborg et al., 1968 medium - IAA Indole-3-acetic acid - MS Murashige and Skoog's (1962) medium     

Plant regeneration from pea protoplasts via somatic embyogenesis

Plant regeneration via somatic embryogenesis was obtained from pea protoplasts. Strong auxins (picloram or 2.4-D) and increased osmolarity of the medium were necessary for embryo induction. Relatively high amounts of embryogenic calli could be obtained in 2 genotypes. After a period on hormone-free medium, a second induction of somatic embryos was possible. Further development of somatic embryos was accomplished on GA₃ — containing medium.Abbreviations ABA abscisic acid - BA 6-benzylaminopurine - 2.4-D 2.4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - Kin kinetin - NAA naphthaleneacetic acid - Pic Picloram, 4-amino-3,5,6-trichloropicolinic acid     

Shoot apex explants for induction of somatic embryogenesis in mature Quercus robur L. trees

A procedure for inducing somatic embryos in shoot apex explants (2 mm) excised from shoot proliferation cultures established from adult oak trees (Quercus robur) was investigated. Embryogenesis was induced in shoot tip as well as leaf explants in three out of the five genotypes evaluated. Somatic embryos were formed by culture in induction medium supplemented with 21.48 μM naphthalene acetic acid and 2.22 μM benzyladenine for 8 weeks, and successive transfer of explants to expression media with a low concentration of growth regulators and without them. Both types of explants formed callus tissue from which somatic embryos developed, indicating indirect embryogenesis. Although the embryogenic frequencies were lower than 12%, it did not prevent the establishment of clonal embryogenic lines maintained by repetitive embryogenesis. Histological study confirmed an indirect somatic embryogenesis process from shoot tip explants, in which leaf primordia and the corresponding axial zones were involved in generating callus, whereas the apical meristem itself did not proliferate. The origin of embryogenic cells appeared to be associated with dedifferentiation of certain parenchymal cells in callus regions after transfer of explants to expression media without auxin. Division of embryogenic cells gave rise to proembryo aggregates of unicellular origin, although a multicellular origin from bulging embryogenic areas would also seem possible. Further development led to the formation of cotyledonary-stage somatic embryos and nodular embryogenic structures that may be considered as anomalous embryos with no clear bipolarity. Inducement of somatic embryos from explants isolated from shoot cultures ensures plant material all year round, thus providing a significant advantage over the use of leaf explants from field-grown trees.     

Effect of copper on shoot and root regeneration in wheat,triticale, rape and tobacco tissue cultures

CuSO₄ (0.1–100 M) significantly enhanced shoot regeneration from calli of wheat and triticale and of tobacco leaf disc cultures. In cultures of wheat and triticale, CuSO₄ also stimulated root formation. When equal concentrations of CuSO₄ were applied in different media, it was found that the components of the basal media had only modifying effects. CuSO₄ pretreatment promoted plant survival when regenerated wheat plants were transferred directly to potting soil. In contrast with CuSO₄, AgNO₃, which also stimulated shoot regeneration, inhibited rooting in wheat and triticale. In Brassica napus callus cultures, AgNO₃ strongly increased morphogenesis, whereas CuSO₄ had no significant effect.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - KN kinetin - NAA naphthaleneacetic acid     

Proliferation and differentiation from endosperms of Carthamus tinctorius

The endosperms of Carthamus tinctorius cv. HUS-305, excised at globular to heart-shaped stages of zygotic embryo development, were cultured on Murashige and Skoog’s medium (MS) supplemented with different concentrations of 6-benzylaminopurine (BAP), kinetin, thidiazuron (TDZ), 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene-acetic acid (NAA). The highest incidence of callusing was on 2,4-D supplemented media. However, embryos differentiated only from the calli developed on media supplemented with BAP, kinetin or TDZ with the last eliciting maximum embryogenic response. The addition of a reduced nitrogen source, casein hydrolysate to MS medium supplemented with BAP and/or NAA, did not stimulate the response. However, adenine sulphate (100 mg dm⁻³) promoted the induction of somatic embryos. Upon transfer to MS basal medium or the same supplemented with 0.61 μM gibberellic acid (GA₃), plumular poles of few embryos elongated resulting in the development of shoots.