Immature Zygotic Embryos Research Articles

Using flow cytometry and cytological analyses to assess the genetic stability of somatic embryo-derived plantlets from embryogenic Musa acuminata Colla (AA) ssp. malaccensis cell suspension cultures

Flow cytometry and chromosome counts were used to analyze the genetic stability of plants regenerated via Musa acuminata Colla (AA) ssp. malaccensis embryogenic cell suspension (ECS) cultures. These cultures were initiated from immature zygotic embryos (IZE) on Murashige and Skoog medium using nine different plant growth regulator (PGR) treatments. Highest percentage of embryogenic calli (EC) formation occurred on media with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D, 97 %), and 8.2 μM of picloram (Pi, 80 %) followed by 2.2 μM 2,4-D (75 %). Embryonic development was synchronized in liquid medium by filtration, and somatic embryo development was achieved with ECS aliquots overlaid on PGR-free medium. The EC medium composition and elapsed time of both short-term (~5 months old) and long-term (~2 years old) ECS cultures influenced plant regeneration, resulting in 65–99 % embryo germination and 50 to 100 % plant conversion. The mean 2C DNA content (1.23 ± 0.002 pg) and chromosome number (2n = 2x = 22) of M. acuminata ssp. malaccensis IZEs, seedlings and sucker plantlets were similar to the reported values. No significant differences were detected among IZEs before culturing, and none were found among the IZEs and leaves of control plants and the plants regenerated from short-term ECS lines when initiated with 2,4-D or Pi vis-a-vis the accession from which they originated. However, plants regenerated from the long-term ECS-L3 culture remained diploid, had the highest DNA content (2C = 1.283 ± 0.01 pg) and were clearly separate from the other regenerated and control plants.

INDUCTION OF DIRECT SOMATIC EMBRYOGENESIS FROM IMMATURE EMBRYOS OF BRASSICA OLERACEA VAR. SABAUDA L.

An efficient plant propagation system through somatic embryogenesis by using immature zygotic embryos was established in Savoy cabbage (Brassica oleracea var. sabauda L.). The effects of zygotic embryo age, presence of 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) (B5-D) in the culture medium, and the pH (5.0 and 5.8) of the B5 plant growth regulator (PGR)-free (B5-0) induction media were investigated. It appeared that the developmental stage of the immature zygotic embryos used as explants was the most important factor for somatic embryogenesis. Highest frequency of somatic embryogenesis (86.67%) was achieved directly on zygotic embryos at cotyledonary stage (1.8 mm long) of development that cultured on B5-0 medium. Optimal pH of the medium was 5.0. Mean number of primary somatic embryos per explant on B5-0 pH 5.0 medium was 7.2. The inclusion of 2,4-D into the induction medium significantly reduced both frequency of somatic embryogenesis (to 53.34 %) and mean number of embryos per explant (to 2.62) in cotyledonary zygotic embryos. After transferring to PGR-free Murashige and Skoog (MS-0) medium 69.69% of the primary somatic embryos of the Savoy cabbage produced secondary somatic embryos (SSEs) Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of Savoy cabbage.

Cultivo in vitro de Embriones Inmaduros en tres Citotipos de Brachyopodium distachyon (L.) P. Beauv.

In vitro culture of inmature embryos in three cytotypes of Brachypodium distachyon (L.) P. Beauv. In this work the in vitro culture response of six population samples of Brachypodium distachyon (two by each one of the cytotypes of 2n = 10 2n = 20; 2n = 30) has been analyzed. Immature zygotic embryos

Assessment of genetic fidelity of somatic embryogenesis regenerated guava (Psidium guajava L.) plants using DNA-based markers

An efficient somatic embryogenesis protocol for plantlet regeneration of the commercially valuable, and nutritionally important, crop guava (Psidium guajava L.) has been established. The immature zygotic embryos were targeted as an explant source for inducing somatic embryogenesis and plant regeneration, using four commercial cultivars (Allahabad Safeda, Lalit, Sardar and Shweta). The plantlets regenerated were assessed for genetic fidelity using random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR) and simple sequence repeat (SSR) molecular markers. A total of 2171 scorable bands were obtained using RAPD, ISSR or SSR, ranging from 300–3500, 250–3000 and 150–280 bp, respectively. No polymorphism was observed among the somatic embryogenesis regenerated plants, compared with respective donor mother plants. The profiles generated based on the three marker systems were found to be highly uniform and approximately 99% bands were monomorphic. This high degree of genetic uniformity (as assessed using the markers) in the somatic embryogenesis regenerated plants indicates genomic stability was maintained through the regeneration protocol. The somatic embryogenesis regenerated plants were hardened and transferred to the field for acclimatization, of which 80% plantlets survived, with all being phenotypically similar to the donor mother plants. We conclude that the RAPD, ISSR and SSR markers were informative and potentially useful in confirming the uniformity assessment of somatic embryogenesis regenerated plants.

Sustainable production of azadirachtin from differentiated in vitro cell lines of neem (Azadirachta indica)

Azadirachtin has high industrial demand due to its immediate application as an ecofriendly, biodegrad- able biopesticide and also due to its various other significant bioactivities. To date, the only commercially feasible way to produce azadirachtin is extraction from seeds, but their availability is very limited as the tree flowers only once a year and only one-third of the fruits are collected due to operational problems. Further, due to the strict out- breeding nature of the plant, the seeds are highly heterozygous, resulting in inconsistent metabolite production. There- fore, in the present study, to achieve sustainable production of azadirachtin, dedifferentiated and redifferentiated calli derived from various explants of neem—zygotic embryo, leaf and ovary—were investigated for their potential to bio- synthesize azadirachtin. High-performance liquid chromatography analysis of theinvitro cell lines showed the presence of azadirachtin in all the samples tested, the content of which in cultured cells varied with explant source and cell dif- ferentiation response. The presence of azadirachtin in samples was further confirmed by positive electrospray ionization mass spectroscopy. The zygotic embryo cultures of neem accumulated much higher amounts of azadirachtin than leaf and ovary cultures. Furthermore, organizedinvitro callus cultures (redifferentiated) supported higher azadirachtin bio- synthesis, while unorganized callus cultures (dedifferentiated) supported the least. The maximum azadirachtin content of 2.33 mg g 21 dry weight was obtained from redifferentiated immature zygotic embryo cultures.

Effect of the genotype, developmental stage and medium composition on the in vitro culture efficiency of immature zygotic embryos from genus Capsicum

A new reliable in vitro technique for immature embryos of Capsicum spp. was developed. A collection of 10 accessions, encompassing the five cultivated species of Capsicum, and four media formulations, combining different levels of sucrose (40g/L vs. 80g/L) and MS salts (½×MS vs. 1×MS), were evaluated. In addition, the four main embryo stages (i.e. globular, heart, torpedo, early cotyledonary) were also considered. Thus, almost 2000 embryos were excised, cultured, and evaluated for germination in the present experiment. Genotype (G), media composition (M), and developmental stage (S) contributed significantly to culture efficiency. G×S and S×M interactions were also significant, although their contribution was lower than individual main factors. C. annuum accession Piquillo, C. frutescens B-144 and, particularly, C. pubescens B-61 showed the highest in vitro germination rates, while C. chinense and C. baccatum accessions showed, in general, the lowest responses. In most cases, the more advanced the embryo stage the higher the culture efficiency. However, for the first time in Capsicum globular embryos from most genotypes were germinated in vitro and also relatively high rates were achieved for heart embryos. Finally, the medium with the lowest levels of both sucrose (40g/L) and MS (½×MS) enabled, in most genotypes and stages of development, the highest in vitro germination rates. In fact, this medium allowed rates of up to 25% in globular embryos. These results provide useful information to those breeders interested on the applications of embryo culture in Capsicum peppers (e.g. shortening breeding cycles, perhaps rescue of interspecific embryos).

High-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis

This study reports high-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis. Numerous green globular structures were directly formed on the surfaces of cotyledons and radicles from 2-week-old immature zygotic embryos at a frequency of 42.1 % when cultured on Murashige and Skoog (MS) medium supplemented with 2 mg l−1 of α-naphthaleneacetic acid (NAA) and 1 mg l−1 of 6-benzyladenine (BA). In comparison, white globular structures and pale-yellow calluses were formed simultaneously at a frequency of 28.3 % when cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The pale-yellow calluses were transferred to MS liquid medium supplemented with 2,4-D to establish embryogenic cell suspension cultures consisting of round, isodiametric cells that formed cell aggregates. Upon plating of these cell aggregates on half-strength MS medium without growth regulators under light conditions, cell aggregates gave rise to numerous globular embryos at a frequency of 56 %. Of the globular embryos, 15 % were successfully converted into cotyledonary embryos when cultured on half-strength MS medium under light conditions. The plant regeneration system of H. cordata established in this study will be useful for the selection, genetic transformation, and mass proliferation of elite clones with medicinal potential.

SOMATIC EMBRYOGENESIS IN SEED LEGUMES: THE POSSIBLE ROLE OF SOLUBLE AUXIN RECEPTORS

ABSTRACT The formation of somatic embryos in higher plants in vitro is an exogenously regulated process which can be divided into (a) the induction phase and (b) the release of induced cells for further development. There are, however, many examples in the literature where induction of somatic embryogenesis is restricted to certain genotypes or cultivars. The solution for overcoming the regeneration problem in these cases requires laborious and time-consuming try-and-error approaches, including the testing of numerous media and growth conditions, with unpredictable success. Since in most plant species, including cereals and seed legumes, the induction of somatic embryogenesis is a strictly auxin-controlled process, we studied the signal transduction pathway, focusing on the primary interaction site, namely auxin recognition. This requires at the beginning the characterization of specific auxin receptors in explants exhibiting embryogenic potential. In immature zygotic pea embryos and shoot tips of etiolat...

In vitro plant regeneration from immature zygotic embryos and repetitive somatic embryogenesis in kohlrabi (Brassica oleracea var. gongylodes)

A simple and efficient protocol for direct somatic embryogenesis and plant regeneration of kohlrabi (Brassica oleracea var. gongylodes) was developed. Somatic embryos were induced from immature zygotic embryos at different developmental stages cultured on Murashige and Skoog medium supplemented with 0, 0.5, 1.0, or 1.5 mg/l 2,4-dichlorophenoxyacetic acid. Zygotic embryos at the early cotyledonary stage, which were cultured for 4 wk on plant growth regulator-free (PGR-free) medium, displayed the highest percentage of somatic embryogenesis (80.7%). Embryogenic tissue could be subcultured on the same medium for over 1 yr. Embryogenic lines derived from early cotyledonary stage zygotic embryos displayed the highest intensity of secondary embryogenesis (highest mean number of new somatic embryos per responsive somatic embryo explant). Histological analyses confirmed the direct origin of the secondary somatic embryos. Prolonged culturing of embryogenic tissue on PGR-free medium led to somatic embryo development into plantlets that were successfully acclimated in the greenhouse with a survival rate of 72.5%. Flow cytometry analysis showed no ploidy variation in 96.7% of the acclimated plants.

Application of embryo culture method in combination with gamma irradiation and ultra sounds: Part I

SUMMARY Immature sunflower ( Helianthus annuus L.) zygotic embryos belonging to R and B sunflower lines were treated with ultra sound and gamma rays before planting in an embryo culture medium. In those circumstances new mutant lines with the altered architecture, increased oil content, 1000-seed weight, number of seed per head, as well as seed yield were developed. Some mutant lines showed very good combining abilities. Line 12004 R (developed trough Embryo culture method in immature zygotic embryos in combination with gamma irradiation at dose 8 Gy), paternal component of hybrid Goryanin, line 12002 R (developed trough Embryo culture method in immature zygotic embryos in combination with gamma irradiation at dose 8 Gy), paternal component of the commercial hybrid Rada and line 12003 R (developed trough Embryo cluture method in immature zygotic embryos in combination with ultra sound at dose 25.5 W/cm 2 for 1 min) and paternal component of the commercial hybrid Yana were realized. Hybrid Goryanin considerably exceeded the average standard (the Bulgarian commercial hybrids Mercury, Perfect and S-205) by seed yield with 15.2%. Hybrid Rada considerably exceeded the average standard of seed yield by 2.3-7.7% (in relation to Bulgarian commercial hybrid Albena and French commercial hybrid Diabolo). The vegetation period was 115 days. The hybrid Yana considerably exceeded the average standard of seed yield by 7.6%-14.4% (in relation to Bulgarian commercial hybrid Perfect and French commercial hybrid Diabolo). Its vegetation period is 110 days. The hybrids Rada and Yana possess the immunity to the parasite Orobanche cumana race G, immunity to Plasmopara helianhi- races 300, 330, 700 and 731, immunity to Phomopsis and Macrophomina , immunity to Sclerotinia sclerotiorum (root form) and tolerance to Phoma.

Development of Transgenic Papaya throughAgrobacterium-Mediated Transformation

Transgenic papaya plants were regenerated from hypocotyls and immature zygotic embryo after cocultivation with Agrobacterium tumefaciens LBA-4404 carrying a binary plasmid vector system containing neomycin phosphotransferase (nptII) gene as the selectable marker and β-glucuronidase (GUS) as the reporter gene. The explants were co-cultivated with Agrobacterium tumefaciens on regeneration medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime for one week. The cocultivated explants were transferred into the final selection medium containing 500 mg/L carbenicillin + 200 mg/L cefotaxime + 50 mg/L kanamycin for callus induction as well as plant regeneration. The callus derived from the hypocotyls of Carica papaya cv. Shahi showed the highest positive GUS activities compared to Carica papaya cv. Ranchi. The transformed callus grew vigorously and formed embryos followed by transgenic plantlets successfully. The result of this study showed that the hypocotyls of C. papaya cv. Shahi and C. papaya cv. Ranchi are better explants for genetic transformation compared to immature embryos. The transformed C. papaya cv. Shahi also showed the maximum number of plant regeneration compared to that of C. papaya cv. Ranchi.

Temporal regulation of somatic embryogenesis in guava (Psidium guajava L.)

SummarySomatic embryogenesis was induced in immature zygotic embryos of guava (Psidium guajava L.) at 10-weeks post anthesis using 2 – 38 d pulses and continuous treatments with different concentrations (0.001 – 3.0 mg l–1) of 2,4-dichlorophenoxyacetic acid (2,4-D). Subsequent development of somatic embryo was observed by sub-culturing onto plant growth regulator (PGR)-free, full-strength Murashige and Skoog (MS) agar medium containing 3% (w/v) sucrose. The maximum responses were shifted gradually from 0.01 mg l–1 2,4-D (continuous or 38 d treatment), to 0.05 mg l–1 2,4-D (28 – 18 d treatment), to 0.1 mg l–1 2,4-D (14 – 12 d treatment) and, finally, to 0.5 mg l–1 2,4-D (10 – 8 d treatment).The highest frequency of embryogenesis (68.8%) and embryogenic intensity (69.2 embryos per explant per culture), as well as moderate frequencies of convertible elongated and short torpedo-stage somatic embryos (15% and 42.9%, respectively), with the highest efficiency (27.5) achieved in the 8 d treatment compared to continuous treatment of zygotic embryo explants with 0.5 mg l–1 2,4-D in MS agar medium containing 3% (w/v) sucrose at both the induction and development phases. Cellular pools of total, free, conjugated, and bound forms of various polyamines (e.g., putrescine, spermidine, and spermine) showed slow increases in the presence of 2,4-D during the 8 d induction phase. This was followed by rapid increases immediately after sub-culturing explant tissue onto the PGR-free MS development medium. The concentrations of various polyamines declined gradually over time on MS development medium. The production, metabolism, and use of different forms of various polyamines at various times (d) after culture initiation correlated with their temporal regulation during the induction and development phases of somatic embryogenesis. Exogenous application of different polyamines during the induction phase resulted in slightly increased frequencies, intensities, and efficiencies of embryogenesis, but with no observable change in the frequencies of the various categories of somatic embryos. The present study indicated that the process of somatic embryogenesis in guava may be less sensitive to PGRs and was induced even in the continuous presence of 2,4-D. Furthermore, somatic embryogenesis in guava was temporally regulated as a function of the exogenous concentration of 2,4-D. In addition, the temporal regulation of somatic embryogenesis was also associated with high levels of production, metabolism, and use of cellular polyamines during the different phases. Hence, improvements in the process of somatic embryogenesis in guava could be regulated by timed applications of 2,4-D alone, or in combination with polyamines, thereby modulating endogenous levels of the latter.

Histological analysis and transcription profiles on somatic embryogenesis in interspecific hybrids of <i>Elaeis guineensis × E. oleifera</i>

Elaeis guineensis (African oil palm) and E. oleifera (American oil palm) are bred to attain high oil yields, disease resistances, and decelerated shoot elongation. We cultivated immature zygotic embryos from backcrossed and F1 interspecific progenies on media containing 110, 150, or 200 mg·l-1 2,4-diclorophenoxyacetic acid (2,4-D) to obtain embryogenic cultures. These were set to multiply on medium containing 8 mg·l-1 2,4-D or lower concentrations of auxins and finally we induced plantlets regeneration, from each zygotic embryo, independently, in order to have the clones organized according to their respective origins. Reductions in auxins induced cultures to revert from highly embryogenic into competent for embryogenesis and finally to organogenic degenerated callus lines. Histology and the expression of SOMATIC EMBRYOGENESIS RECEPTOR KINASE, DEHYDRIN, DEFENSIN, TRANSPOSASE, and LIPID TRANSFER PROTEIN were analyzed on four callus lines representative of morphological aspects consistently observed. The highest number of embryogenic cultures was obtained on 150 mg·l-1 2,4-D. Maturation and multiplication of somatic embryos through secondary embryogenesis occurred simultaneously on 8 mg·l-1 2,4-D. LIPID TRANSFER PROTEIN expression was detected in one of the embryogenic cultures and correlated with protoderm onset. Three six-week cycles on induction medium yielded 1.5 shoots above 6 cm per poly-embryogenic complex, which performed better than embryoids individualized mechanically. Rooting was observed for 77% and 82% of shoots from these two types of explants, respectively. Rooted plantlets ready for acclimatization were obtained nine months after shoot induc

Polyethylene glycol effects on somatic embryogenesis of papaya hybrid UENF/CALIMAN 01 seeds

Among commercial fruits, papaya (Carica papaya L.) is notably one of the most common ones around the world. The aim of the current study was to establish a somatic embryogenesis protocol for the hybrid UENF/CALIMAN 01, elucidating the effects of sucrose and 2,4-dichlorophenoxyacetic acid (2,4-D) on the induction of embryogenic cultures and the effect of polyethylene glycol (PEG) on somatic embryo maturation. Immature zygotic embryos of C. papaya hybrid UENF/CALIMAN 01 were cultured in MS culture medium supplemented with different concentrations of sucrose (30 and 60 g L-1) and 2,4-D (0, 20, 40 and 80 μM). The combination of 30 g L-1 sucrose and 20 μM 2,4-D resulted in the highest induction rates and the largest callus diameters. Furthermore, this combination was associated with the greatest potential to form somatic embryos. To promote maturation, cultures were inoculated in MS medium and exposed to different concentrations of PEG (0, 30 and 60 g L-1). The 60 g L-1 PEG treatment group had the highest average for the number, maturation and conversion of somatic embryos that resulted in plantlets at the end of the process. The addition of PEG to MS medium led to increased protein synthesis. These results are unprecedented for C. papaya hybrid UENF/CALIMAN 01 as the current study demonstrates the development of somatic embryogenesis in this high quality hybrid.

Recurrent somatic embryogenesis and plant regeneration from immature zygotic embryos of cabbage (Brassica oleracea var. capitata) and cauliflower (Brassica oleracea var. botrytis)

A simple and rapid protocol was established for repetitive somatic embryogenesis and subsequent plant regeneration in two important Brassica oleracea varieties, cabbage and cauliflower. Direct regeneration of somatic embryos (SEs) was achieved from immature zygotic embryos cultured on B5 plant growth regulator (PGR)-free (B5-0) induction medium and on B5 medium supplemented with 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) (B5-D). Zygotic embryos of both cabbage and cauliflower at the cotyledonary (C) stage (1.8 mm long) incubated on B5-0 medium displayed the highest embryo-forming capacities (EFCs) of 11.84 and 11.95, respectively. Secondary somatic embryos (SSEs) appeared on the cabbage and cauliflower’s primary embryos at a high frequency (83.3 and 87.5 %, respectively), and this process continued in a repetitive way on PGR-free Murashige and Skoog (MS-0) medium. The embryogenic potential of the cultures with a gradual diminution was maintained for 10 months (ten cycles). A total of 20 % of the mature SSEs from cabbage and 55 % from cauliflower spontaneously regenerated plantlets on MS-0 medium. The addition of 1 mg l−1 6-benzyladenine (BA) or 6-furfurylaminopurine (Kin) in the regeneration medium significantly improved somatic embryo conversion into plantlets by up to 56 % in cabbage and 79 % in cauliflower. Regenerated plants acclimated successfully to ex vitro conditions and displayed morphological and reproductive characteristics similar to seed-derived plants. Effective recurrent somatic embryogenesis may be an appropriate practical solution for clonal propagation and genetic modifications of cabbage and cauliflower.

Agrobacterium-mediated genetic transformation of selected tropical inbred and hybrid maize (Zea mays L.) lines

The study was carried out to evaluate the amenability of tropical inbred and hybrid maize lines, using Agrobacterium mediated transformation technique. Agrobacteriumtumefaciens strains EHA101 harbouring a pTF102 binary vector, EHA101, AGL1, and LBA4404 harbouring pBECK2000.4 plasmid, LBA4404, GV and EHA105 harbouring pCAMBIA2301 plasmid, and AGL1 harbouring the pSB223 plasmid were used. Delivery of transgenes into plant tissues was assessed using transient β-glucuronidase (gus) activity on the 3rd and 4th day of co-cultivation of the infected Immature Zygotic Embryos (IZEs) and embryogenic callus. Transient gus expression was influenced by the co-cultivation period, maize genotype and Agrobacterium strain. The expression was highest after the 3rd day of co-culture compared to the 4th day with intense blue staining was detected for IZEs which were infected with Agrobacterium strains EHA105 harbouring pCAMBIA2301 and EHA101 harbouring pTF102 vector. Putative transformants (To) were regenerated from bialaphos resistant callus. Differences were detected on the number of putative transformants regenerated among the maize lines. Polymerase chain reaction (PCR) amplification of Phosphinothricin acetyltransferase (bar) and gus gene confirmed the transfer of the transgenes into the maize cells. Southern blot hybridization confirmed stable integration of gus into PTL02 maize genome and segregation analysis confirmed the inheritance of the gus. A transformation efficiency of 1.4 % was achieved. This transformation system can be used to introduce genes of interest into tropical maize lines for genetic improvement.

Tracheary elements that resemble secondary xylem in calli derived from the conifers, Torreya nucifera and Cryptomeria japonica

Differentiated cells were recognized in calli derived from needles of Torreya nucifera and in calli derived from immature zygotic embryos of Cryptomeria japonica. Some differentiated cells resembled tracheary elements of primary xylem with spiral or reticulate thickening of cell walls. Other cells resembled tracheary elements with thick cell walls and bordered pits, which are features of secondary xylem. These tracheary elements were formed in cell clusters. Tracheary elements in calli of T. nucifera formed more highly developed structures, such as bordered pits and spiral thickening, than those of C. japonica. Cultured cells derived from conifers might provide a good model for studies of the differentiation of secondary xylem in vitro.

Plant regeneration through somatic embryogenesis from immature and mature zygotic embryos of Musa acuminata ssp. burmannica

A simple and efficient protocol has been developed for in vitro regeneration of M. acuminata ssp. burmannica (AA) plants. Somatic embryos were produced when immature and mature zygotic embryo explants were cultured on Murashige and Skoog medium supplemented with plant growth regulators 2,4-dichlorophenoxyacetic acid; (2,4-D), picloram or benzyl adenine and indole acetic acid. In general, immature embryos responded better than mature embryos. Callus proliferation was highest in medium supplemented with 2,4-D (4.5 μM). Subsequent transfer of callus to fresh medium produced rapidly proliferating embryogenic calli. Embryogenic calli were maintained in complete darkness for 15 d followed by cycles of 8 h dark and 16 h light, under white fluorescent lamps with a light intensity of 3,000 lm/m2 and at temperature of 28 ± 2°C. Regeneration of embryogenic calli into plantlets was higher for immature embryos (76.6%) than for mature embryos (50.6%). This plant regeneration protocol using mature or immature zygotic embryos, via somatic embryogenesis, has significant potential to improve germination efficiencies of hybrid progenies used in conventional breeding strategies. Furthermore, tests on seed storage showed that seed viability rapidly decline after harvesting and was negligible after 9 mo of storage. This indicates using freshly harvested seeds as explant material is necessary for maximizing the tissue culture response.

In vitro germination and embryogenic competence acquisition of Euterpe edulis Martius immature zygotic embryos

This study evaluated different aspects of in vitro germination and embryogenic competence of immature zygotic embryos of E. edulis. The embryos germinated on full or half-strength MS (MS or MS/2) medium combined with sucrose (20, 30 and 40 g L-1). The effect of calcium chloride concentrations (0, 2, 4, 8, and 12 mM) on the induction of somatic embryogenesis was tested. The embryos were germinated on MS or MS/2. Germination of zygotic embryos and the number of roots per plantlet were not affected by the culture medium and sucrose concentration. Plantlet height and fresh weight were influenced by both; the difference was greatest in MS medium with 40 g L-1 sucrose. The induction of somatic embryogenesis was not influenced by doses of calcium chloride, whereas the number of somatic embryos formed was affected. The germination capacity of somatic embryos of heart-of-palm tree was not influenced by the media tested.

In vitro pollination in poplar of section Populus

Two methods of in vitro pollination of aspen were tested and compared, first with moist pollen by transferring swollen pollen grains from a wet agar surface to the stigma and, second, using dry pollen grains. In vitro pollination with dry pollen grains appeared to be more efficient and this method was applied in three subsequent years. Additionally, pollination was performed with selected pollen grains in order to obtain triploid plants. Diploid pollen was induced by heat-treatment of pollen mother cells, but the enrichment of unreduced pollen from natural pollen mixtures was also successful. The selection of diploid pollen, which is larger in diameter than haploid pollen, was performed by sieving using micro sieves. Five triploid plants from different poplar combinations were obtained from 1,227 zygotic embryos cultured after in vitro pollination of 2,676 ovaries with dry pollen over 3 years. Embryo rescue was used to facilitate the development of immature zygotic embryos. The results of this study demonstrate the possibility to effect pollination using selected single pollen grains with the help of in vitro techniques.