Repetitive Somatic Embryogenesis Research Articles

Repetitive somatic embryogenesis and plant regeneration in Garcinia indica choiss

Immature seeds of Garcinia indica Choiss, were exeised from immature fruits and cultured on Lloyd and McCown (1980), woody plant medium (WPM) with different combinations of auxins and cytokinins. Somatic embryos were obtained on the media supplemented with 6-benzy laminopurine (BA; 2.2–22.1 μM) alone or in combination with α-naphthalene acetic acid (NAA; 2.6 μM) with 80% frequency within a period of 2–3 wk. Subculture of embryos on medium containing BA (16.0 μM) supplemented with indole-3-acetic acid (IAA: 2.8–5.7 μM) and/or kinetin (4.6 μM) gave rise to clusters of secondary somatic embryos along with maturation of primary embryos. In subsequent subculture on hormone-free half-strength WPM, the embryo clusters germinated with an increase in the number of secondary somatic embryos. About 70% of somatic embryos germinated into complete plantlets, which were successfully established under greenhouse conditions.

IN VITRO MORPHOGENESIS OF FIVE ORCHIDS

IN VITRO MORPHOGENESIS OF FIVE ORCHIDS

Repetitive Somatic Embryogenesis of Ocotea catharinensis Mez. (Lauraceae): Effect of Somatic Embryo Developmental Stage and Dehydration

Repetitive embryogenesis of Ocotea catharinensis from globular/early cotyledonary somatic embryos was successfully supported by WPM supplemented with 22.7 g l−1 sorbitol, 20 g l−1 sucrose, 400 mg l−1 glutamine and 2 g l−1 Phytagel. The best medium to induce repetitive embryogenesis in cotyledonary somatic embryos was half strength WPM supplemented with 20 g l−1 sucrose, 400 mg l−1 glutamine, 1.5 g l−1 activated charcoal and 2 g l−1 Phytagel. The mature somatic embryos gradually air dehydrated showed repetitive embryogenesis after subculture on half strength B5 medium supplemented with 20 g l− sucrose, 20 g l−1 Phytagel, 1.5 g l−1 activated charcoal, 115.6 µM gibberellic acid and 214.8 µM naphthaleneacetic acid. The early cotyledonary, cotyledonary and mature somatic embryos tolerated respectively 95, 86 and 54% fresh weight losses without losing their repetitive embryogenesis potential. Cotyledonary and mature somatic embryos gradually air dehydrated in sealed Petri dishes showed 40–41% repetitive embryogenesis respectively after 20 days and 12 weeks desiccation storage. Repetitive embryogenesis in cotyledonary somatic embryos was significantly stimulated by chemical dehydration with 0.5 M sorbitol and 56% repetitive embryogenesis was achieved even after exposure to 2 M sorbitol for 24 h. The cotyledonary somatic embryos when alginate-encapsulated showed 47% repetitive embryogenesis even after chemical dehydration in 1.5 M sorbitol for 4 days followed by 1 h air dehydration, but failed to survive to the same dehydration conditions without encapsulation. The optimized repetitive embryogenesis and desiccation protocols offer the possibility to use in vitro techniques for continuous reliable somatic embryo production and short term germplasm storage.

In vitro somatic embryogenesis in turf‐type bermudagrass: roles of abscisic acid and gibberellic acid, and occurrence of secondary somatic embryogenesis

AbstractBermudagrass is an important warm‐season turfgrass species that is recalcitrant in regeneration in tissue culture. In a previous report, we observed that somatic embryogenesis of immature inflorescence culture was substantially improved when low levels of 2,4‐dichloro‐phenoxy acetic acid (1 mg/l) and 6‐benzylaminopurine (BAP, 0.01 mg/l) were included in the callus induction medium. The object of this study was to further improve the culture conditions to enhance somatic embryo formation and plantlet regeneration. It was shown that the abscisic acid supplement (2 or 5 mg/l) to the above callus induction medium further enhanced somatic embryogenesis in hybrid bermudagrass (Cynodon dactylon×Cynodon transvaalensis) cv. ‘Tifgreen’. The addition of gibberellic acid (0. 2 mg/l) to the BAP (1 mg/l)‐containing regeneration medium accelerated germination/regeneration of the somatic embryos. Secondary and repetitive somatic embryogenesis, which is rarely reported in monocots, was observed in common bermudagrass (Cynodon dactylon, cv. ‘Savannah’), and a full course of such a development was captured by a periodical microphotography. Scanning electron microscopy further confirmed the observation.

Somatic embryogenesis in perennial statice Limonium bellidifolium, Plumbaginaceae

Cotyledon explants from perennial statice Limonium bellidifolium (Statice caspia Willd.) were cultured on Murashige and Skoog's basal medium (MS) supplemented with various levels of 2,4-D, kinetin and sucrose. Embryogenic calluses developed over a period of 10 days with the highest response at 4.5 μM (1 mg l−1) 2,4-D, 0.5 μM (0.1 mg l−1) kinetin and 117 mM sucrose. Following induction, the calluses were transferred to MS media supplemented with 88 or 117 mM sucrose and 0 or 0.5 μM kinetin. Somatic embryos at the globular, heart-shaped, torpedo, and cotyledonary stages developed. Fully germinated plantlets developed with the best response in medium supplemented with 117 mM sucrose and 0.5 μM kinetin. Direct somatic embryogenesis without a callus phase was observed with some of the cotyledon explants. Induction, maturation and germination of somatic embryos on the optimized media were equally effective using cotyledon, hypocotyl and root explants. Serial sections of L. bellidifolium cotyledon explants cultured for two weeks indicated that pro-embryogenic masses originated from parenchyma cells below the epidermis. Further histological observations of embryogenic calluses confirmed the initiation and development of globular and heart-shaped embryos and repetitive somatic embryogenesis. Ultrastructural observations indicated that the embryogenic cells were less vacuolate with abundant organelles compared to the cells of the explant. This is the first report of somatic embryogenesis in the Plumbaginaceae.

High-yielding repetitive somatic embryogenesis and plant recovery in a selected tea clone, 'TRI-2025', by temporary immersion.

Methods for improving the efficiency of repetitive somatic embryogenesis and plant recovery from somatic embryos of clonal tea, TRI 2025 [Camellia sinensis (L.) O. Kuntze] were investigated by optimising the immersion frequencies of the explants using a modified temporary immersion system (TIS). The relative efficiencies of three conventional methods for multiplying embryos were compared with the temporary immersion method. The highest rate of multiplication of secondary embryos (24-fold) was achieved using the TIS. By controlling the immersion cycles, we achieved more consistent, synchronised multiplication and embryo development with a high level of plant recovery. A one-step computer-programmed immersion protocol based on a single, simple medium with no growth regulators was developed, enabling multiplication, maturation, germination and plant recovery within 17 weeks. Plantlets recovered through this method were hardy, with 2- to 5-cm-long shoots containing a minimum of 2-4 lush green leaves and a well-formed taproot. Callus formation, hyperhydricity and other developmental abnormalities were not observed at any stage in the process. Plantlets produced using this method were successfully acclimatised to glasshouse conditions. This protocol avoids culture transfers, and thus minimises the risk of contamination and reduces labour costs. This technique could have significant commercial implications in tea propagation as it has the potential for large-scale production with considerably reduced production costs.

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.

Repetitive somatic embryogenesis in Medicago truncatula ssp. Narbonensis and M. truncatula Gaertn cv. Jemalong

Medicago truncatula ssp Narbonensis and four genotypes of M. truncatula Gaertn cv. Jemalong were tested for their somatic embryogenesis potential using a two-step protocol. In the first step, embryogenic callus was induced in folioles isolated from shoots grown in vitro and cultured on Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid and zeatin. In the second step, somatic embryos were allowed to develop from the induced callus in MS growth-regulator-free medium. Individual somatic embryos were then isolated and transferred again to growth regulator free medium where they formed secondary somatic embryos in repetitive cycles. Conversion of somatic embryos into plantlets was achieved by isolating late-torpedo-phase somatic embryos with distinct cotyledons and reculturing them onto MS growth regulator free medium. The system of repetitive somatic embryogenesis in M. truncatula described here represents a permanent source of embryogenic material that can be used for the genetic modification of this species.

In vitro regeneration of onion through repetitive somatic embryogenesis

A reliable protocol for the regeneration of onion through repetitive somatic embryogenesis was established. Embryogenic callus was derived from mature seeds on Murashige and Skoog (MS) medium supplemented with 2 mg dm-3 2,4-dichlorophenoxyacetic acid (2,4-D). Somatic embryos aroused on the surface of calli cultures and formed plantlets after the removal of 2,4-D or its substitution with 1 mg dm-3 kinetin (Kin). Reculturing the somatic embryos on 2,4-D containing medium led to secondary embryos formation. The embryogenic cultures which were preserved for five months on maintenance medium containing 2 mg dm-3 2,4-D + 0.5 mg dm-3 Kin have retained their ability for regeneration, while those kept on 2,4-D only, failed to form plantlets. Electrophoretic analysis of total soluble proteins revealed that the competence for successful conversion of somatic embryos into plantlets is associated with the expression of new set of proteins (112, 58 and 30 kD). The regenerated plants were successfully transferred to the soil.

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

Genetically transformed alfalfa (Medicago sativa L., cv. Zajecarska 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 β-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.

Repetitive somatic embryogenesis in peanut cotyledon cultures by continual exposure to 2,4-d

Somatic embryos from immature cotyledons in peanut (Arachis hypogaea) were initiated on media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-d). Over 90% primary embryogenesis and 41–46% repetitive embryogenesis were obtained 12 weeks after initiation by maintaining embryogenic cultures on medium containing 20 mg 1-1 2,4-d. Maintenance of cultures on medium with 30 or 40 mg I-1 2,4-d resulted in lower primary and secondary embryogenesis, and proliferation of nonembryogenic callus. Transfer of embryogenic cultures to a secondary medium with 10 or 20 mg I-1 2,4-d significantly enhanced secondary embryogenesis compared to basal medium without the growth regulator. The use of Murashige & Skoog versus Finer's media had no significant effect on embryogenesis (85–95%), repetitive embryogenesis (11–37%) or mean embryo number. Secondary embryogenesis was also maintained for over one year by repeated subculture of isolated somatic embryos on medium with 20 mg I-1 2,4-d.

The effect of auxin type and concentration on pecan (Carya illinoinensis) somatic embryo morphology and subsequent conversion into plants

Embryogenic cultures were initiated from immature pecan zygotic embryos. Explants were induced for one week on Woody Plant Medium with either α-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid at 2, 6 or 12 mg/l, then subcultured monthly to fresh basal medium. Observations were made on callus production, embryo formation, and embryo morphology. Somatic embryo morphology and overall callus proliferation were affected by auxin type. Callus proliferation was less extensive and more somatic embryos resembling zygotic embryos were obtained from cultures initiated with α-naphthaleneacetic acid than with 2,4-dichlorophenoxyacetic acid. Repetitive somatic embryogenesis was obtained in all auxin treatments. Conversion into plantlets was affected by somatic embryo morphology in that embryos with poorly developed apices exhibited lower percentages of conversion than those with well developed single or multiple apices. Consequently, although more embryos were obtained with 2,4-dichlorophenoxyacetic acid, naphthaleneacetic acid was the superior auxin for production of somatic embryos more likely to convert into plants.

A continuous culture system of direct somatic embryogenesis in microspore-derived embryos of Brassica juncea

An efficient culture system has been developed for repeated cycles of somatic embryogenesis in microspore-derived embryos of Brassica juncea without a callus phase. Haploid embryos produced through anther culture showed a high propensity for direct production of somatic embryos in response to 2 mgL(-1) BA and 0.1 mgL(-1) NAA. The embryogenic cultures which comprised the elongated embryonal axis of microspore-derived embryos when explanted and grown on the medium of same composition produced a large number of secondary embryos. These somatic embryos in turn underwent axis elongation and produced more somatic embryos when explanted and cultured. This cycle of repetitive somatic embryogenesis continued with undiminished vigour passage after passage and was monitored for more than a year. Somatic embryos from any passage when isolated at cotyledonary stage and grown on auxin-free medium for 5 days and then on a medium containing NAA (0.1 mgL(-1)), developed into complete plants with a profuse root system and were easily established in the soil. The cytology of the root tips of these plants confirmed their haploid nature. The total absence of callus phase makes the system ideal for continuous cloning of androgenic lines, Agrobacterium-mediated transformation and mutation induction studies.

Repetitive somatic embryogenesis and plant recovery in white clover

Breeding and selection was used to generate a population of white clover (Trifolium repens L.) from cultivar Osceola with a high embryogenic capacity. Somatic embryos were obtained from immature cotyledons of white clover placed onto EC6 basal medium containing 40 mg L(-1) of 2,4-D and 6% sucrose. The effects of 2,4-D at 20 and 40 mg L(-1) and of the carbohydrates, sucrose and maltose, were evaluated for their influence in the establishment of repetitive somatic embryogenesis. To determine the optimal protocol for plant recovery from somatic embryos, the effects of MS vs. EC6 basal salts, sucrose vs. maltose, B5 vitamins vs. yeast extract, and inclusion or exclusion of activated charcoal were evaluated. Repeated subculture of white clover somatic embryos on EC6 basal medium containing 6% sucrose with 2,4-D at 20 or 40 mg L(-1) effectively maintains repetitive embryogenesis. Medium containing MS salts with 6% maltose as the carbohydrate source was the most efficient for plant recovery.

Repetitive somatic embryogenesis from peanut cultures in liquid medium

A regeneration system based on repetitive somatic embryogenesis was developed for peanut (Arachis hypogaea L.). Embryogenic suspension cultures were initiated using individual somatic embryos induced from immature cotyledons cultured on a modified Murashige and Skoog medium containing 40 mg/l 2,4-D for 30 days. After transfer to a modified MS liquid medium, the somatic embryos produced masses of secondary and tertiary embryos which continued to proliferate following manual separation and subculture of the embryogenic clumps. The cultures exhibited exponential growth, and have been maintained for over one year without apparent loss of embryogenic potential. Further embryo development, germination, and conversion were achieved by placing embryo clumps onto hormone-free, solid medium. The inclusion of a desiccation period during embryo development enhanced conversion four-fold. Plants have been established in soil and appear to be phenotypically normal.

Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut, Juglans regia L. cv Manregian

Plants were regenerated by somatic embryogenesis from endosperm tissue of open-pollinated seeds of Juglans regia L. cv Manregian. These plants were obtained by growing endosperm tissue on media similar to those used for plant regeneration from walnut cotyledons (Tulecke and McGranahan 1985). The plants appear morphologically uniform and have a triploid chromosome number of 3n=48. Nine plants have been grown to a young sapling stage in soil. This embryogenic line from endosperm has been maintained in culture for two years by the process of repetitive somatic embryogenesis.