Isozyme patterns in zygotic and somatic embryogenesis of carrot

Isozyme patterns of carrot (Daucus carota L.) zygotic embryos between the torpedo stage up to 5-day-old seedlings have been compared with those of the similar stages from the embryogenic cell suspension culture to the late somatic plantlet. Somatic embryos blocked at the torpedo stage by -cyclodextrine have also been analyzed. All these stages have been analyzed with respect to seven different enzyme systems: arylesterase, glucosephosphate isomerase, phosphogluconate dehydrogenase, alcohol dehydrogenase, isocitrate dehydrogenase, aspartate aminotransferase and phosphoglucomutase (EC 2.7.5.1, PGM). The relationships between the different stages of both types of embryogenesis have been visualized using an unrooted tree. Generally, profiles of somatic embryos were different from those of zygotic embryos. Interestingly however, a typical zygotic embryo pattern was found in the cyclodextrine-blocked somatic embryos. Only aspartate aminotransferase patterns revealed a similarity between zygotic and somatic torpedo embryos. Both plantlet types showed close patterns with common isozymes. Moreover, similarities were evident between somatic plantlets and cell suspensions. A few isozymes appeared to be stage specific markers: esterase 10–11 were specific to achenes and early germination, phosphogluconate dehydrogenase 8 was specific to 4–5 day-old seedlings and phosphoglucomutase 1 and 7 and alcohol dehydrogenase 4 were markers for zygotic embryos. No somatic embryogenesis specific isozyme could be found. We show that patterns can be associated with particular tissue formation: mainly, aspartate aminotransferase 2 and 1, phosphoglucomutase 8 and 9 and phosphogluconate dehydrogenase 7 coincided with apical meristem initiation and phosphoglucomutase 4 and 5, zones b and d of esterase and zone b of phosphogluconate dehydrogenase coincided with vascular bundle formation.Abbreviations ADH alcohol dehydrogenase - CD -cyclodextrine - CS cell suspension culture - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraaeetie acid - LiBo lithium hydroxide/boric acid - PEG polyethylene glycol - PVP polyvinylpyrrolidone - SEg somatic embryo at the globular stage - SEh heart stage - SEte early torpedo stage - SEtl late torpedo stage - SEce early cotyledonary stage - SEcl late cotyledonary stage - SECD somatic embryo blocked at the torpedo stage with -cyclodextrine - EST esterase - GOT aspartate aminotransferase - IDH isocitrate dehydrogenase - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) - PMS phenazonium methosulfate - PGD phosphogluconate dehydrogenase - PGI glucosephosphate isomerase - PGM phosphoglucomutase - SO dry seed - S1–3 seed after 1–3 days of germination - SP1–2 young and old somatic plantlets - ZE zygotic embryo - ZP4–5 4–5 day-old seedlings     

Genotypic control of peanut somatic embryogenesis

The protocol for obtaining a high frequency of plant development via somatic embryogenesis from mature zygotic embryo-derived leaflets of peanut (Arachis hypogaea L.) involves multiple stages; these include the induction of embryogenic masses, development of embryos, radicle emergence/conversion of embryos and the development of plants from rooted abnormal embryos. Sixteen genotypes were subjected to this protocol by exposing mature zygotic embryo-derived leaflets to the common media sequence and comparing responses. Although the protocol was effective for all the genotypes, variation in frequency of response at each stage of development indicated that, with the exception of root meristem differentiation and subsequent radicle emergence, the whole process of somatic embryogenesis depended on the genotypic constitution of the original plant. The failure of somatic embryos to undergo conversion to plantlets could be a genotype-dependent characteristic. Received: 5 June 1997 / Revision received: 2 December 1997 / Accepted: 12 December 1997     

Propagation of Lirlodendron hybrids via somatic embryogenesis

Propagation of hybrids between the Chinese tuliptree (Liriodendron chinense) and the North American yellow-poplar (Liriodendron tulipiferea) could be greatly accelerated with a highly productive somatic embryogenesis system. Flowers were collected from a single Chinese tuliptree and the anthers used for controlled pollinations of 4 yellow-poplar mother trees. Aggregates of samaras resulting from the pollinations were harvested 8 weeks post-pollination. Following surface disinfestation, samaras were dissected and embryos and endosperm were cultured together on a semisolid induction medium containing 9.0 M 2,4-dichlorophenoxyacetic acid and 1.1 M benzyladenine. Following 2–3 months on induction medium, an average of 15.6 percent of the explants produced either somatic embryos or proembryogenic masses. Compared to pure yellow-poplar embryogenic cultures, putative hybrid cultures tended not to maintain growth as proembryogenic masses while exposed to auxin, instead proliferating via repetitive embryogenesis as globular-stage embryos. Four to six weeks following transfer of globular embryos to basal medium, mature embryos were produced from the putative hybrid lines. Mature embryos germinated following transfer to basal medium lacking casein hydrolysate. Plantlets survived transfer to potting mix and acclimatization to greenhouse conditions. Verification of the hybrid nature of the embryogenic lines and somatic embryo-derived plantlets was accomplished by Southern hybridization analysis with a species-specific DNA marker.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - PEM proembryogenic mass - CH casein hydrolysate - RFLP restricted fragment length polymorphism - CTAB hexadecyltrimethylammonium bromide - TBE Tris-borate-EDTA - SDS sodium dodecylsufate - SSC sodium citrate/chloride     

High frequency somatic embryogenesis in Coffea canephora

Leaf explants of Coffea canephora (P. ex Fr.) produced a friable yellow callus when they were cultured on a conditioning basal medium with 2.2 M 2,4-D, 2.4 M IBA and 9.8 M 2iP for 4 weeks then on an induction basal medium with 4.4 M 2,4-D and 17.8 M BA for 10 weeks. This calus could be maintained by means of regular subcultures or it could give rise to somatic embryos depending on the culture medium. Cytological studies documented somatic embryogenesis and embryo development.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - 2iP 2-isopentenyladenine - MS Murashige & Skoog medium - NAA -naphthaleneacetic acid - NPR nucleoplasmic ratio - PGR plant growth regulator     

Organogenesis and somatic embryogenesis in Cupressus sempervirens

Adventitious buds of Cupressus sempervirens L., were formed on excised mature embryos cultured for 10 days on half-strength Quoirin and Lepoivre medium (1/2QP) with 10 M N⁶-benzyladenine. For shoot development, embryos were transferred to 1/2QP without growth regulators. Axillary shoot formation and rooting occurred spontaneously as adventitious shoots aged and transfer intervals were increased. Embryogenic tissue was obtained from immature embryos on induction media consisting of von Arnold and Eriksson (AE) or Gupta and Durzan (DCR) salts with 10 or 20 M 2,4-dichlorophenoxyacetic acid. Cultures were maintained on DCR with 5 M -naphthaleneacetic acid and 5 M BA.Abbreviations BA N⁶-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - ABA absicic acid - AC activated charcoal - NAA -naphthaleneacetic acid - QP Quoirin & Lepoivre (1977) - AE von Arnold & Eriksson (1981) - DCR Gupta & Durzan (1985)     

Direct somatic embryogenesis fromBegonia gracilis explants

Direct somatic embryogenesis ofBegonia gracilis was achieved from microcultured laminar segments and petioles on Murashige and Skoog medium with 0.5 mg 1^–1 kinetin and 2% coconut water. Somatic embryos were obtained with greater frequency from petiole explants than from leaf blade sections. Under red light (45 mol m^–2 s^–1), approximately 80% of the petiole explants successfully produced somatic embryos but only 30% of the leaf blade sections responded. However, somatic embryos were significantly more abundant on responding lamina explants (60–70 embryos/leaf section) than on petioles (40–50 embryos/petiole). These trends were similar for explants kept in the dark, but overall production was lower. Somatic embryos were produced more quickly (5 weeks) from petioles than from lamina explants (8 weeks). The somatic embryos germinated to produce plantlets and subsequently shoot cultures with the same appearance as the parental clone.Abbreviations BA benzyladenine - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid - SE somatic embryo     

High frequency somatic embryogenesis from coffee leaves

An improved procedure for the induction, proliferation and regeneration of embryogenic callus from coffee leaf explants has been developed. The optimal culture conditions for callus induction and somatic embryogenesis yielded so-called high frequency embryogenic callus ofCoffea canephora P. ex Fr., Arabusta and Congusta, more rapidly and abundantly than other published procedures.Coffea arabica L. genotypes, however, were less responsive to the procedure. The highest multiplication rate of embryogenic callus in liquid culture, which avoided the differentiation of embryos, was obtained by culture at an inoculum density of 10 g callus 1^-1 in a modified MS medium containing 4.5 M 2,4-dichlorophenoxyacetic acid, under 3 mol m^-2 s^-1 illumination, and subcultured every 7–10 days. The best long-term maintenance of embryogenic potential was obtained by culture of aggregates (250–1000 m in diameter) at an inoculum density of 5 g 1^-1, with medium renewed every 3–4 weeks. Under these conditions, embryogenic potential ofC. canephora callus was maintained for over 2 years. Analysis of nutrients absorbed by the callus cultures demonstrated that half strength MS macro- and micro-salts were not depleted during at least 3 weeks of sustained culture. The highest regeneration of embryogenic callus required the omission of 2,4-D and a reduced culture density of 1 g 1^-1. Under these conditions of culture, 1 g ofC. canephora or Arabusta callus produced 1.2 and 0.9×10⁵ somatic embryos, respectively, after 8–10 weeks in liquid regeneration medium. This was an overall reduction of 4–6 months from explant to regenerant, when compared with other procedures.Abbreviations BA N⁶-benzyladenine - HFSE high frequency somatic embryogenesis - IAA indole-3-acetic acid - IBA indole-3-butyric acid - rpm rotations per minute - LFSE low frequency somatic embryogenesis - MS Murashige & Skoog medium - PPF photosynthetic photon flux - 2,4-D 2,4-dichlorophenoxyacetic acid - 2-iP 2-isopentenyladenine     

Soybean somatic embryogenesis: Effects of hormones and culture manipulations

Somatic embryos were induced in cultures of immature soybean (Glycine max (L.) Merr) embryos, or isolated cotyledons on MS modified medium supplemented with NAA and 2,4-D, BAP and ABA. When NAA and 2,4-D were compared at similar concentrations (25 and 23 M), 2,4-D produced larger number of somatic embryos, however, embryogenesis efficiency was improved in media containing NAA by using higher levels (100–150 M) of the auxin. Somatic embryo morphology varied with auxin type: NAA-induced embryos more closely resembled zygotic embryos than did 2,4-D-induced embryos. Additions of BAP or ABA to auxin-containing media had either no effect or reduced embryo production, although ABA altered the morphology of 2,4-D-induced embryos. In media containing both NAA and 2,4-D, the latter was dominant in terms of embryo morphology. The effects of subculture frequency and of transfers between 2,4-D and NAA media were investigated: Subculture frequency influenced mainly the frequency of normal embryos, while preculture on 2,4-D increased subsequent embryogenesis efficiency on NAA medium but reduced the frequency of normal embryos.Abbreviations Em^-2 s^-1 microEinsteins per square meter per second - NAA -naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - ABA abscisic acid - BAP benzylamino purine This paper (No. 86-3-96), is published with the approval of the director of the Kentucky Agricultural Experiment Station.     

Plant regeneration via somatic embryogenesis in ginger

Embryogenic callus cultures of ginger were induced from young leaf segments taken from in vitro shoot cultures. Among the four auxins tested in Murashige & Skoog medium, dicamba at 2.7 M was most effective in inducing and maintaining embryogenic cultures. Efficient plant regeneration was achieved when embryogenic cultures were transferred to Murashige & Skoog medium containing 8.9 M benzyladenine. Histological studies revealed various stages of somatic embryogenesis characteristic of the monocot system. The in vitro-raised plants have been established in soil.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid     

Regeneration of Acacia mangium through somatic embryogenesis

 Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from immature zygotic embryos of Acacia mangium. Embryogenic callus was induced on MS medium containing combinations of TDZ (1–2 mg/l), IAA (0.25–2 mg/l) and a mixture of amino acids. Globular embryos developed on embryogenic callus cultured on the induction medium. Nearly 42% of embryogenic cultures with globular embryos produced torpedo- and cotyledonary-stage embryos by a two-step maturation phase. The first stage occurred on 1/2-strength MS basal medium containing 30 g/l sucrose and 5 mg/l GA₃ followed by the second stage on 1/2-strength MS basal medium containing 50 g/l sucrose. Of the cotyledonary-stage somatic embryos, 11% germinated into seedlings that could be successfully transferred to pots. Light- and scanning electron microscopy showed that the somatic embryos originated from single cells of the embryogenic callus. Further, a single cell layer could be detected beneath the developing somatic embryos that appeared to be a demarcation layer isolating the somatic proembryonic structure from the rest of the maternal callus. A suspensor-like structure connected the globular embryos to the demarcation layer. This is the first successful report of plant regeneration through somatic embryogenesis for this economically important tropical forest species. Received: 20 January 2000 / Revision received: 28 September 2000 / Accepted: 29 September     

Potential of somatic embryogenesis in Prunus avium immature zygotic embryos

For the purpose of developing somatic embryogenesis in Prunus avium L., immature zygotic embryos, collected from five donor trees and sorted into two size classes (C1: 2.5–3.5 and C2: 3.6–4.5 mm), received various experimental treatments. When cultured for 10 days on an inductive medium containing 18.1 M 2,4-dichlorophenoxyacetic acid (2,4-d) and 9.3 M kinetin, then transferred to fresh medium without growth regulators, 2.5% of the C1 class cotyledons expressed direct somatic embryogenesis. C2 class cotyledons were less responsive. The response was also influenced by the chosen donor tree. In a few cases, spontaneous germination occurred. The presence of a root meristem was clearly demonstrated by histological examination of longitudinal sections. The replacement of half the amount of 2,4-d, present in the inductive medium mentioned above, by the same quantity of naphthaleneacetic acid reduced the incidence of somatic embryogenesis. Conversely, a rhizogenic response was strongly enhanced. When submitted to an inductive medium containing indoleacetic acid and zeatin without any subcultures for 3 months, C1 class cotyledons were the most morphogenic and developed leaves and cotyledon-like structures.Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - IBA indolebutyric acid - NAA -naphthaleneacetic acid     

Improvement of somatic embryogenesis in highland-papaya cell suspensions

Axillary buds (2 mm) from 3-year-old Carica pubescens Lenné et Koch (highland papaya) fruit-bearing plants grown in the greenhouse were cultivated in NN-medium supplemented with different growth regulators naphthaleneacetic acid and indoleacetic acid in combination with Zeatin, benzyladenine, Kinetin and thidiazuron. Several responses were observed within 2–3 months; namely, sprouting of the preformed axillary buds, bud branching into multiple shoots, callus formation at the basal end of the explant and somatic embryogenesis in the preformed callus. Somatic embryogenesis was frequent in most of the tested growth regulator combinations, with the exception of thidiazuron which showed no effect. A much higher yield of somatic embryos could be obtained in suspensions. Somatic embryogenesis was enhanced by the occurence of adventive embryogenesis on single embryos as globular embryo clusters. This was observed in cell suspensions initially grown in a WPM-medium with 2,4-dichlorophenoxyacetic acid, or in combination with benzyladenine or zeatin, for 6 days, then maintained in a growth regulator-free medium under continuous agitation (50 RPM) on an orbital shaker for 3 months. Single cells grown in the absence of 2,4-dichlorophenoxyacetic acid did not initiate embryogenesis and de-differentiated into callus. Plantlets were recovered after transfer of mature embryos from cell suspensions into Magenta flasks. In a second subculture, adventitious embryogenesis occurred spontaneously in clusters at the globular embryo stage under the same growth conditions, yielding a high number of embryos. The culture conditions described above allowed initiation of a large number of somatic embryos directly from cell suspensions through adventive somatic embryogenesis and indirectly from callus on axillary buds.Abbreviations 2,4-d dichlorophenoxyacetic acid - CH casein enzymatic hydrolysate - BA benzyladenine - FAA formalin:acetic acid:alcohol - Glu l-glutamine - IAA indoleacetic acid - NAA naphthaleneacetic acid - NN Nitsch and Nitsch-medium (1969) - TDZ thidiazuron - SD standard deviation     

Callus induction and somatic embryogenesis of Phalaenopsis

Callus induction and plant regeneration through somatic embryogenesis in Phalaenopsis Richard Shaffer `Santa Cruz' were examined. Protocorm-like body (PLB) segments formed calli in Vacin and Went medium with sucrose. The optimal concentration of sucrose was 40 g ⋅ l^–1. Medium containing 200 ml ⋅ l^–1 coconut water together with 40 g ⋅ l^–1 sucrose was effective for callus induction. Gellan gum was suitable than agar as a gelling agent for callus induction. The calli easily formed PLBs after being transferred to a medium without sucrose. Histological observation suggested that the PLBs were somatic embryos. No variation was observed in the flowering plants regenerated through somatic embryogenesis. Received: 11 June 1997 / Revision received: 6 October 1997 / Accepted: 18 October 1997     

Rapid and repetitive plant regeneration in sweetpotato via somatic embryogenesis

An efficient in vitro plant regeneration system characterized by rapid and continuous production of somatic embryos using leaf and petiole expiants has been developed in sweetpotato [Ipomoea batatas L. (Lam.)]. The optimal somatic embryogenic response was obtained in the genotype PI 318846-3 with a two-step protocol: (1) stage I-incubation of expiants in the dark for 2 weeks on Murashige Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) (2.5 mg/l) and 6-benzylaminopurine (0.25 mg/l) and, (2) stage II-culture in the light on MS medium with abscisic acid (ABA) (2.5 mg/l). The addition of ABA was critical for enhanced production of somatic embryos. Secondary somatic embryos were produced from the primary embryos cultured on MS medium with 2,4-D at 0.2 mg/l. The somatic embryos were converted into normal plantlets when cultured on basal MS medium. Upon transfer to soil, plants grew well and appeared normal with no mortality. The system of somatic embryogenesis described here will facilitate tissue culture, germplasm conservation and gene transfer research of sweetpotato due to its rapidity (6 to 10 weeks), prolific plant production by direct embryogenesis, ease of secondary somatic embryo production and reproducibility.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine, 2,4-D-2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - KIN kinetin - MS medium of Murashige and Skoog (1962) - NAA 1-naph-thaleneacetic acid - PIC picolinic acid - TDZ thidiazuron     

A novel type of somatic embryogenesis in Coffea arabica

A novel type of somatic embryogenesis characterized by an efficient and highly synchronized embryo formation was observed in embryogenic callus of Coffea arabica initiated on Murashige and Skoog medium containing kinetin (4 mg/l) and 2,4-D (1 mg/l). It occurs in suspension and goes along with the suppression of High Frequency Somatic Embryo Induction (HFSE). This is achieved by favoring during cultivation senescence-or necrosis-like processes which apparently do not impair the competence for embryogenesis. Since the resulting embryos germinate at a rate of 94.5 % without the need of a maturation step, we propose the term Self-Controlled Somatic Embryogenesis (SCSE).In addition, HFSE was optimized using half-strength liquid medium with 0.1 mg/l kinetin and 0.25 mg/l 2,4-D for proliferation of embryonic tissue, and 2.6 mg/l ABA for maturation of embryos. Yields as well as germination rates of HFSE embryos were markedly lower as compared to SCSE.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - NAA 1-naphthaleneacetic acid - HFSE high frequency somatic embryo induction - LFSE low frequency somatic embryo induction - SCSE self-controlled somatic embryogenesis     

Effects of light quality on somatic embryogenesis in Araujia sericifera

The effects of photoperiod, light quality and end-of-day (EOD) phytochrome photoconversion on somatic embryogenesis (SE) of Araujia sericifera petals have been studied. Petals from immature flowers were cultured under 8- and 16-h photoperiods using Gro-lux fluorescent lamps. The photon fluence rate was 90–100 μmol m⁻² s⁻¹ and the red (R):far-red (FR) ratio was 98. R, FR, R followed by FR (R-FR) and FR followed by R (FR-R) light treatments were applied for 3 weeks at the end of the photoperiods. In a set of experiments, dl - α -difluoromethylarginine (DFMA) or methylglyoxal bis (guanylhydrazone) (MGBG), both inhibitors of polyamine biosynthesis, were added to the culture medium in order to study the involvement of polyamine metabolism. The level of SE was the same in long (LD) and short (SD) days. Thus, the light effect was accomplished after 8 h. All EOD treatments that decreased the P_fr level inhibited SE when applied after SD, but not after LD. The FR-R treatment after LD caused an additional stimulatory effect on SE, even in the presence of polyamine inhibitors. DFMA inhibited SE in both SD and LD, but MGBG did not modify SE in either SD or LD. The R, FR and R-FR treatments did not alter the level of SE when applied after LD in the presence of DFMA or MGBG. However, these treatments decreased SE after SD when the medium contained polyamine inhibitors. Our results suggest that Gro-lux lamps, which produce an extremely high R:FR ratio, promote SE in A. sericifera and a timing response to phytochrome photoconversion during photoperiodic induction. Thus, our data corroborate the involvement of phytochromes and polyamines in SE in A. sericifera, which responded as a light-dominant long-day plant.     

A protocol for repetitive somatic embryogenesis from mature peanut epicotyls

 The effects of 11 different auxins and one cytokinin-like compound were tested at four concentrations for their ability to induce primary and repetitive somatic embryos from mature, dry peanut (Arachis hypogaea L.) epicotyls of genotype AT120. Treatment with picloram and centrophenoxine at 83.0 and 124.4 μm resulted in the greatest number of embryos per explant and the highest percentage of explants responding. In a follow-up experiment, picloram, centrophenoxine, and dicamba were tested at 83.0 and 124.4 μm on four peanut genotypes (AT120, 59-4144, GK7, and VC1). Picloram and centrophenoxine induced similar numbers of globular-stage and total embryos from each genotype, while dicamba was less effective. Similar results were observed with percentage of responding axes. Genotypes AT120 and VC1 yielded more clusters of repetitive embryos than GK7 and 59-4144. After 5 months, embryos derived from repetitive embryogenic cultures were converted into mature plants. Received: 8 February 1999 / Revision received: 9 June 1999 / Accepted: 30 June 1999     

Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

Genetically transformed alfalfa (Medicago sativa L., cv. Zajearska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l^–1 sucrose, 1 g l^–1 yeast extract and 0.05 mg l^–1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l^–1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or -glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - Km kanamycin - NPTII neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid - BM basal medium     

High frequency of plant regeneration in sunflower from cotyledons via somatic embryogenesis

Summary A plant regeneration methodvia somatic embryogenesis of severalHelianthus annuus L. genotypes was developed. Starting from cotyledonary explants high frequency embryo induction was obtained following several subcultures on defined media. An appropriate cotyledon developmental stage was identified. Etiolated explants and darkness treatment were necessary to obtain somatic embryos in all tested genotypes. After 20–25 days on somatic induction medium containing an auxin:cytokinin ratio of 1:1, the germination of embryos was induced by a reduction of the hormonal ratio (1:2). Shoots were excised from callus and transferred onto a medium containing various vitamins. The range of embryogenesis frequency was 33–72%, depending on the genotype. High frequency of rooting (49–82%) was obtained using a medium supplemented with 0.5 mg/L of ancymidol and by a reduction of photoperiod. A large percentage of somatic embryos developed into normal regenerated plants producing viable seeds.Abbreviations MS Murashige and Skoog (1962) - NAA naphthaleneacetic acid - BAP benzylaminopurine - GA₃ gibberellic acid - EIM embryo induction medium - GM germination medium - VM vitamins medium - RA2 ancymidol rooting medium - EtOH ethanol