Conversion Of Somatic Embryos Research Articles

Effects of proliferation, maturation, and desiccation methods on conversion of soybean somatic embryos

Cermination of soybean [Glycine max (L.) Merrill] somatic embryos and conversion to whole plants are generally low. This study was conducted to investigate the effects of proliferation, maturation, and desiccation methods on conversion of soybean somatic embryos to plants. Soybean cv. Jack somatic embryos, proliferated on a solid medium containing 90.5 μM (20 mgl−1) 2.4-dichlorophenoxyacetic acid (2.4-D) (MSD20), showed a regeneration rate signficantly higher than those proliferated in a liquid medium containing 45.25 μM (10mgl−1) 2,4-D (FN Lite). When a liquid medium without 2,4-D and B5 vitamins (FN Superlite) was used for maturation, the duration of time necessary for embryo development could be shortened by more than a month compared to maturation on a standard solid medium (MSM6AC). An air-drying method, in which somatic embryos were desiccated in an empty sealed Petri dish for 3–5d, gave rise to the best germination efficiency among the four desiccation methods tested: fast, slow, air, and KCl methods. The final percentage of moisture seems important since embyros over-dried by the fast and slow methods did not convert well into plants.

Proliferation and maintenance of embryogenic capacity in elm embryogenic cultures

This paper investigates maintenance and proliferation of somatic embryogenesis systems for Ulmus minor and U. glabra. Proliferation occurred with subculture of embryogenic calluses. The calluses were mainly formed by friable nodules composed of meristematic cells organized into proembryogenic cell masses (PEMs) and thin-walled vacuolated parenchymatic cells. Cotyledonary embryos, with procambial strands and differentiation of their vascular tissues as well as visible root meristems, were identifiable after 18d of culture on a proliferation medium with 0.44 μM benzyladenine (BA). The shoot meristem was only occasionally well developed. Somatic embryo multiplication from elm embryogenic calluses is a clearly asynchronic system, and PEMs as well as embryos at all stages of development are observed simultaneously at the end of subculture period. Factors affecting the proliferation of elm embryogenic callus, such as culture medium, carbon source and genotype, were studied. Basal medium (MS) or medium supplemented with 0.44 μM BA produced the highest number of somatic embryos. Somatic embryo production was higher with sucrose or glucose than with maltose, and significant differences were also found among the four embryogenic lines tested. The use of liquid medium with filter paper support is an essential step for the survival of isolated somatic embryos during the germination stage. The addition of 0.22 μM BA′ to liquid MS medium was the best treatment for germination and plantlet conversion of elm somatic embryos.

Vegetative propagation of Quercus suber L. by somatic embryogenesis

The regeneration of somatic seedlings from selected 100-year-old cork oak trees is reported. The induction of somatic embryogenesis from leaves of epicormic shoots was significantly affected by genotype, harvesting time and their interaction. Leaves from all five selected trees produced somatic embryos when the segments of branches used as sources of epicormic shoots were collected in May. Genotype, but not the level of photosynthetically active radiation, affected the proliferation of the embryogenic lines and the number of detachable embryos that could be obtained from them. Genotype also affected several steps leading to conversion of somatic embryos, from germination to complete acclimatisation of somatic seedlings. Almost 40% of the somatic embryos from all lines germinated, showing coordinated root and shoot growth. Although the mean percentage of recovery for the whole process was low, plants could be regenerated from four of the five trees tested.

Maturation and germination of oak somatic embryos originated from leaf and stem explants: RAPD markers for genetic analysis of regenerants

Experiments were performed to determine the influence of maturation medium carbohydrate content on the rates of germination and plantlet conversion (root and shoot growth) of somatic embryos from four embryogenic lines derived from leaf or internode explants of Quercus robur L. seedlings. The conversion rate was favoured by high carbohydrate content as long as the maturation medium contained at least 2% sucrose, which was necessary for healthy embryo development. Given this, sorbitol and mannitol favoured the conversion rate more efficiently than sucrose, the highest rate, 32%, being achieved by medium with 6% sorbitol and 3% sucrose. Maturation treatment did not affect the root or shoot lengths of converted embryos. In supplementary experiments, 2 weeks of gibberellic acid treatment between maturation and germination treatments did not improve germination rates, but did reduce root length and the number of leaves per regenerated plantlet. In the four embryogenic lines tested, plant recovery rate was enhanced by inclusion of benzyladenine into the germination medium following culture of the embryos on maturation medium with 6% sorbitol and 2-3% sucrose. In embryogenic systems it is important to assess the uniformity of the regenerants. Random amplified polymorphic DNA (RAPD) analysis using 32 arbitrary oligonucleotide primers was performed to study variability in DNA sequences within and between four embryogenic lines. No intraclonal nor interclonal polymorphism was detected between embryogenic lines originating from different types of explant from the same seedling, but every one of the primers detected enough polymorphism among clones originating from different plants to allow these three origins to be distinguished. No differences in DNA sequences between regenerated plantlets and their somatic embryos of origin were detected, but a nodular callus line that had lost its embryogenic capacity was found to be mutant with respect to three other clones originating from the same plantlet. This study shows that high carbohydrate levels in the maturation medium significantly increase plant conversion of oak somatic embryos, which exhibit no variation in DNA sequences when proliferated by secondary embryogenesis.

Comparison of the effects of kanamycin and geneticin on regeneration of papaya from root tissue

Kanamycin and geneticin are commonly used for the selection of neomycin phosphotransferase II (npt II) transformed plants. Since papaya tissue is sensitive to both antibiotics, it is difficult to explore their effects on the regeneration process solely based on using non-transformed tissues. Adventitious roots derived from npt II-transgenic and non-transgenic papaya shoots in vitro were used as explants in this investigation. The effects of kanamycin and geneticin on callus formation, embryogenesis, and conversion of somatic embryos to shoots were compared. Callus growth derived from npt II-transformed root explants was apparently enhanced on kanmycin within 50–200 mg l−1 or on geneticin within 12.5–50 mg l−1 as compared to those on antibiotic-free controls. The percentages of npt II-transformed somatic embryo-forming callus were not significantly different (16.3–18.3%) on geneticin less than 6.25 mg l−1 and only slightly reduced (11.2–15.7%) on geneticin within 12.5–50 mg l−1, whereas, formation of somatic embryos was strongly suppressed on kanamycin media. Conversion rates of npt II-transformed somatic embryos to shoots were not significantly different among all kanamycin or geneticin treatments. Percentages of the callus derived from non-transformed root explants were greatly reduced on the medium containing more than 25 mg l−1 kanamycin or geneticin, and no somatic embryos formed from untransformed callus on any kanamycin or geneticin media. Our results indicated that somatic embryogenesis of callus derived from npt II-transformed root explants of papaya was strongly inhibited by kanamycin. Thus, to regenerate npt II-transformed cells from papaya root tissue, we recommend using the lower concentration geneticin (12.5–25 mg l−1) to avoid the adverse effects of kanamycin on embryogenesis.

Somatic embryogenesis and plant regeneration from cotyledon explants of a timber-yielding leguminous tree, Dalbergia sissoo Roxb.

Efficient plant regeneration through somatic embryogenesis was achieved from callus cultures derived from semi-mature cotyledon explants of Dalbergia sissoo Roxb., a timber-yielding leguminous tree. Somatic embryos developed over the surface of embryogenic callus and occasionally, directly from cotyledon explants without intervening callus phase. Callus cultures were initiated from cotyledon pieces of D. sissoo on Murashige and Skoog (1962) medium supplemented with 4.52, 9.04, 13.57, and 18.09 mumol/L 2,4-dichlorophenoxyacetic acid and 0.46 mumol/L Kinetin. Maximum percentage response for callus formation was 89% on MS medium supplemented with 9.04 mumol/L 2,4-D' and 0.46 mumol/L Kn. Somatic embryogenesis was achieved after transfer of embryogenic callus clumps to 1/2-MS medium without plant growth regulators (1/2-MSO). Average numbers of somatic embryos per callus clump was 26.5 on 1/2-MSO medium after 15 weeks of culture. Addition of 0.68 mmol/L L-glutamine to 1/2-MSO medium enhanced somatic embryogenesis frequency from 55% to 66% and the number of somatic embryos per callus clump from 26.5 to 31.1. Histological studies were carried out to observe various developmental stages of somatic embryos. About 50% of somatic embryos converted into plantlets on 1/2-MSO medium containing 2% sucrose, after 20 days of culture. Transfer of somatic embryos to 1/29-MSO medium containing 10% sucrose for 15 days prior to transfer on 1/2-MS medium with 2% sucrose enhanced the conversion of somatic embryos into plantlets from 50 to 75%. The plantlets with shoots and roots were transferred to 1/2 and 1/4-liquid MS medium, each for 10 days, and then to plastic pots containing autoclaved peat moss and compost mixture (1:1). 70% of the plantiets survived after 10 weeks of transfer to pots. 120 regenerated plantlets out of 150 were successfully acclimatised. After successful acclimatisation, plants were transferred to earthen pots.

Somatic embryogenesis from zygotic embryos of Euterpe oleracea Mart.

The aim of this work was to study the morphogenetic responses of zygotic embryos of açai palm (Euterpe oleracea Mart.) submitted to several conditions of in vitro culture. Several research experiments were conducted, in laboratory, using vegetable material collected from açai palm plants at Embrapa Amazon Oriental, Belém-PA, Brazil. It was possible to verify the expression of a direct, repetitive and no-synchronized model of somatic embryogenesis in mature zygotic embryos cultivated in primary MS medium supplemented with 2,4-D (339.36 muM) and transferred to a secondary MS medium in the presence of NAA (0.537 muM) and 2iP (12.30 muM). The conversion of somatic embryos into seedlings was reached after 210 days with the transfer of the cultures to a third medium with sucrose and mineral salts concentrations reduced to a half, without growth regulators.

Maturation and Conversion of Somatic Embryos of Three Genetically Diverse Rose Cultivars

Embryogenic callus cultures of three genetically diverse cultivars of rose (Rosa hybrida L.), the floribunda `Trumpeter', the multiflora `Dr. Huey', and the hybrid tea `Tineké', were used to study the effect of various carbohydrates and osmotically active compounds on somatic embryo maturation and conversion. Cotyledonary-stage embryos were produced by dispersing callus in liquid medium followed by filtration to isolate globular-stage embryos. Quantitative experiments were conducted to determine maturation and conversion of the three rose cultivars in response to medium with sucrose, glucose, fructose, or maltose as the primary carbon source and also in response to various concentrations of either myo-inositol, polyethylene glycol, or mannitol in combination with 3% sucrose. Conversion of 27% was achieved for `Trumpeter' embryos following their maturation on 3% fructose. `Dr. Huey' embryos required maturation on medium containing 3% sucrose supplemented with either 2.5% or 5% mannitol for 36% and 61% conversion, respectively. Maturation of `Tineké' embryos on either 3% sucrose, 3% glucose, or 3% fructose resulted in a maximum 12% conversion.

Somatic embryogenesis and plant regeneration in Eucalyptus globulus Labill

Somatic embryogenesis was induced from juvenile explants of Eucalyptus globulus Labill. Mature zygotic embryos, isolated cotyledons, hypocotyls, leaves and stems were cultivated at 24°C in darkness on Murashige and Skoog medium supplemented with 3% (w/v) sucrose and different growth regulator combinations. Callus was formed at the surface of the explant in all tested media containing sucrose but not in those containing mannitol. Calli were transferred to the same medium without growth regulators (MSWH) after 25 days. Somatic embryogenesis was observed in callus derived from cotyledon explants and from entire mature zygotic embryos in the presence of 3–15 mg l–1 α-naphthalene acetic acid (NAA) alone or in the presence of 1 mg l–1 NAA combined with 1 mg l–1 2,4-dichlorophenoxyacetic acid. Best embryogenic rates were obtained in the presence of 3–5 mg/l NAA, as approximately 30% of callus formed on these media produced somatic embryos. Exposure, for >1 week, to the highest NAA concentrations tested (15 mg l–1) failed to induce somatic embryogenesis. Addition of 500 mg l–1 casein hydrolysate and 500 mg l–1 glutamine to the induction medium increased the number of abnormal somatic embryos. Conversion of somatic embryos to plantlets (21%) was obtained when they were transferred to medium free of growth-regulators.

Direct induction of somatic embryogenesis from immature zygotic embryo of cashewnut ( Anacardium occidentale L.)

Direct somatic embryogenesis was induced in precociously germinated immature zygotic embryos (IZEs) of cashewnut ( Anacardium occidentale L.). The IZEs were cultured on semisolid Murashige and Skoog (MS) basal medium with 0.5% activated charcoal and incubated in dark. After 4–5 weeks, somatic embryos were induced directly from the radical tip of IZEs on medium with 2,4-dichlorophenoxyacetic acid (2,4-D) (5 μM)+ BAP (5 μM)+ gibberellic acid (GA 3) (3 μM) with 10% frequency of embryogenic response, which was 9% when 2,4-D (5 μM) was used alone or with Kinetin (Kin) (5 μM). The somatic embryos were transferred to medium with abscissic acid (ABA) (20 μM) and maltose (3%) for further maturation and, studies are being carried out for conversion of somatic embryos into plantlets. A histological study of somatic embryo development was carried out where it was seen that the somatic embryos arose from the outermost cell layer of radical tip.

Ameliorative Effect of Cyanobacterial Extract on the Conversion of Somatic Embryos to Plantlets in Banana

Abstract The conversion of somatic embryos into plants of banana cv. Rasthali (AAB) was studied using different concentrations of cyanobacterial extracts (CYE) from Anabaena strain L-31 and Anacystis nidulans strain BD-1. Extracts from both the strains stimulated embryo to plantlet conversion with varying frequencies. L-31 extract showed maximum conversion frequency (58% at 20% CYE) followed by BD-1 (54% at 50% CYE) and control (45-46% at 0% CYE). Plantlet development as seen by root and shoot growth was also promoted in the presence of CYE. These results will be useful in optimization of methods towards the conversion frequency of somatic embryos into plantlets.

Direct somatic embryogenesis from mature embryos of sandalwood

Plants were regenerated from mature zygotic embryos of sandalwood (Santalum album L.) through direct somatic embryogenesis. Somatic embryos were formed directly without any intervening callus phase on zygotic embryos plated on Murashige and Skoog (MS) medium containing thidiazuron or benzylaminopurine. Individual somatic embryos were then isolated and transferred to MS medium without cytokinin on which they formed secondary embryos in repetitive cycles with or without the addition of indole acetic acid to the medium. Conversion of somatic embryos into plantlets was achieved by isolating somatic embryos with distinct cotyledons and reculturing them onto half-strength MS medium with GA3 (1.4 μM). Recovered plantlets were acclimatised and grown in the greenhouse. This is the first report on in vitro regeneration via direct somatic embryogenesis of sandalwood.

Colchicine, trifluralin, and oryzalin promoted development of somatic embryos in Ilex paraguariensis (Aquifoliaceae)

A protocol for somatic embryogenesis and plant regeneration of Ilexparaguariensis St. Hil. from embryos cultures was developed. Heart stage zygotic embryos were removed from seeds of immature, light green fruit and treated with antimicrotubule agents (0.1; 0.2, and 0.5% colchicine for 24 and 48 h; 1; 10, and 20 μM of either trifluralin, ααα- trifluoro- 2,6-dinitro-N,N- dipropyl-p-toluidine, or oryzalin, 3,5-dinitro-N4, N-dipropylsulphate during 48 h). The embryos were cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar (1/4MS), and 0.46 μM zeatin. Cultures were incubated in darkness at 27 ± 2 °C. All thetreatments provoked a diminution of the number of germinated embryos and in some of the treated embryos somatic embryogenesis was induced. Somatic embryo maturation and conversion into whole plants could be achieved by culturing the embryos on 1/4MS lacking hormones and incubated at 27 ± 2 °C, 14 h photoperiod (116 μmol m-2s-1). Mostof the plants regenerated from somatic embryos appeared morphologically normaland grew under greenhouse conditions. Only 2 plants out of 152 studied contained the tetraploid number of the chromosomes (2n = 4x = 80), meanwhile the rest of the plants had the normal diploid number of chromosomes (2n =2x = 40). Somatic embryos with abnormal morphology were also observed.

Somatic embryogenesis in cell suspension cultures of Acacia sinuata (Lour.) Merr.

Suspension cultures initiated from calluses derived from seedling leaf explants of Acacia sinuata (Lour.) Merr. produced somatic embryos. Embryogenic callus was induced on semisolid MS (Murashige and Skoog, 1962) medium supplemented with 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.22 μM 6-benzylaminopurine. A high frequency of somatic embryos was induced in MS liquid medium supplemented with 4.52 μM 2,4-D and 10% coconut water. Further studies on ontogeny of somatic embryos showed that the cells destined to become somatic embryos divided into spherical proembryos. Subsequent development led to the formation of globular, heart, torpedo-shaped and cotyledonary-stage embryos. The conversion of somatic embryos occurred on auxin-free MS medium. Effects of various auxins, cytokinins, carbohydrates and amino acids in enhancing productin, of somatic embryos were studied. MS medium supplemented with 87.64 mM sucrose and 342.46 μM glutamine promoted higher somatic embryo production whereas cytokinin had no effect and led to recallusing of embryos. About 8–10% of embryos converted into plants.

Somatic embryogenesis and embryo encapsulation in Fraxinus angustifolia Vhal.

SummaryThis is the first paper on somatic embryogenesis in F. angustifolia, an important tree in the Mediterranean Basin for landscaping and wood production. The induction of somatic embryogenesis from immature zygotic embyros and the subsequent development, maturation, encapsulation and conversion of somatic embryos into plantlets, as well as the long-term maintenance of embryogénie capacity, are reported. The developmental stage of immature zygotic embryos affected morphogenic response. An auxin-cytokinin ratio of 2:1 (2,4-dichlorophenoxyacetic acid: benzyladenine) was essential for induction. A combination of 2,4-dichlorphenoxyacetic acid (0.44 |j,M) and benzyladenine (4.4 |j,M) has made it possible to maintain embryogénie capacity for over three years. Mannitol (30 g 1_1) and activated charcoal (2 g l”1) positively affected somatic embryo maturation. The encapsulation of somatic embryos in sodium-alginate beads adversely affected plant conversion, regardless of nutrient additions to the alginate. Sixty-five per cent of non-encapsulated somatic embryos were successfully converted into whole plantlets on hormone-free MS medium in comparison to 23% of the encapsulated ones.

Effect of polyethylene glycol and sugar alcohols on soybean somatic embryo germination and conversion

Polyethylene glycol 4000, mannitol and sorbitol were tested as supplements to a liquid Finer and Nagasawa medium-based histodifferentiation/maturation medium, FNL0S3, for soybean (Glycine max L. Merrill) somatic embryos of ‘Jack’ and F138 or ‘Fayette’. Significant differences were found among types and levels of osmotica for their influence on quantity of mature embryos recovered, and on germination (root and shoot emergence) and conversion of embryos into plants. Supplementation of FNL0S3 with 5% polyethylene glycol or 1.5% sorbitol improved germination frequencies without limiting embryo histodifferentiation. Supplementation with 3% sorbitol resulted in a 9-fold increase in germination frequencies and a 13-fold increase in conversion frequencies of ‘Fayette’ and ‘Jack’ embryos. However, these improvements were accompanied by a significant, 22% reduction in fresh weight of mature embryos. Overall, 3% sorbitol was found to be the best of the osmotic supplements tested, resulting in 51 5% conversion frequency (mean SE), as compared to 4 1% in the control. Supplementation of FNL0S3 with 3% mannitol did not improve embryo maturation. Abbreviations: FN ‐ Finer and Nagasawa (1988); H/M ‐ histodifferentiation/maturation; MS ‐ Murashige and Skoog (1962); PEG ‐ polyethylene glycol

The effectiveness of nitrogen sources in Feijoa somatic embryogenesis

Immature and mature zygotic embryos excised from Feijoa fruits were employed as explants and the effects of NH4 + and NO3 − ionic concentration in basal LPm culture medium supplemented with 2,4-D (10 μM) were evaluated. Moreover, the addition of 4 mM of Asn, Gln, and Arg, and levels of Gln (0 to 8 mM) were tested. The original NH4 + and NO3 − concentration present in the LPm culture medium supplemented with Gln (4 mM) resulted in the highest somatic embryo number from immature zygotic embryos. For mature zygotic embryos, the addition of Asn, Gln or Arg to the basal LPm culture medium resulted in improved somatic embryogenesis induction. Ten weeks in culture allowed the highest somatic embryo number when mature zygotic embryos were used as explant. Half-strength MS culture medium supplemented with BAP (0.5 μM) enhanced the conversion of somatic embryos to plantlets.

Somatic embryogenesis from embryogenic cell suspension cultures of california poppy, Eschscholzia californica Cham

A somatic embryogenesis protocol was developed for Eschscholzia californica Chan. (California poppy) using embryogenic cell suspensions and optimized media conditions. Rapidly-growing, finely-dispersed embryogenic cell suspension cultures were established from embryogenic callus and maintained in B5 liquid media supplemented with 0.5 mg 1−1 (2.26 μM) 2,4-dichlorophenoxyacetic acid. Culture conditions were optimized by investigating the effect of basal media composition, gyratory shaker speed, various carbon sources, different cytokinins, and AgNO3 on the efficiency of somatic embryogenesis. After 40 d in culture, the somatic embryos that formed were counted and their overall growth expressed as pecked cell volume. The selected media consisted of either Gamborg (B5) or Murashige and Skoog (MS) salts and vitamins supplemented with 40 g 1−1 (117 mM) sucrose, 0.05 mg 1−1 (0.22 μM) 6-benzylaminopurine, and 10 mg l−1 (58.8 μM) AgNO3. Somatic embryo production was substantially reduced at shaker speeds above 40 rpm. Glucose and snerose were the most effective carbon sources, whereas fructose, galactose, and maltose resulted in a reduced yield and growth of somatic embryos. The development of somatic embryos was promoted by AgNO3 at concentrations below 10 mg l−1 (58.8 μM). A semi-solid medium containing 1.5 g l−1 Gel-rite produced the highest frequency of somatic embryo conversion, and promoted the efficient growth of plantlets. Using the reported protocol, over 500 viable somatic embryos were produced per 25 ml of embryogenic cell suspension culture.

PROPAGAÇÃO VEGETATIVA DA GUABIROBEIRA (Campomanesia xanthocarpa BERG.) IN VITRO E POR ESTAQUIA

A guabirobeira (Campomanesia xanthocarpa Berg.) é uma Mirtácea frutífera lenhosa nativa da região Sul do Brasil, cuja exploração comercial pode ser fomentada por estudos sobre sua propagação vegetativa. Foram empregadas diversas técnicas de propagação vegetativa com o objetivo de estudar o comportamento da espécie. O trabalho está dividido em duas fases: estaquia e micropropagação. Para a estaquia semi-lenhosa foram testados 2 tipos de substrato (100% Plantmax; 2/3 Plantmax + 1/3 areia) e 7 tratamentos (água; ETANOL 50%; ETANOL 20%; ETANOL 40%; AIB 500 mg.L-1; AIB 1.000 mg.L-1; AIB 2.000 mg.L-1). Para a estaquia herbácea foram testados 6 tratamentos (testemunha, água; ETANOL 50%; AIB 1.000 mg.L-1; AIB 5.000 mg.L-1; AIB 10.000 mg.L-1) e 2 tempos de imersão (30 s e 2 h). Não houve formação de raízes em nenhum dos experimentos. Na micropropagação a fonte de explantes foram plantas em casa de vegetação e plantas adultas a campo. Em todos os experimentos a desinfestação foi baseada em etanol e NaOCl comercial e o meio de cultura foi o MS. Os experimentos com micro-estacas de campo ou casa de vegetação não passaram da fase de desinfestação, devido à contaminação bacteriana endógena. A micro-estaquia de plântulas assépticas utilizou explantes de sementes germinadas in vitro a partir de sementes retiradas de frutos maduros ou iniciando a maturação. A desinfestação foi realizada sobre os frutos ou sobre as sementes já separadas da polpa e ambas as formas foram eficientes. O teste de multiplicação mostrou que o meio sem reguladores de crescimento apresentou brotações mais longas, enquanto o meio acrescido de 0,5 mg.L-1 de BAP apresentou maior número de brotações por estaca. Houve formação de raiz em meio sem reguladores de crescimento. Na cultura de meristemas foram utilizados meristemas apicais e axilares. Os resultados demonstraram ser possível a eliminação da contaminação bacteriana, mas não houve desenvolvimento e multiplicação dos meristemas. A embriogênese somática foi induzida sobre embriões zigóticos desenvolvidos, retirados de frutos em fase inicial de maturação. Foram realizados testes com diversas combinações de reguladores de crescimento (2,4-D, ANA, AIB, BAP e 2i-P) em várias concentrações, visando otimizar as fases da embriogênese, da indução à conversão. Na fase de indução a formação de embriões somáticos ocorreu nos meios com 0,5 a 1 mg.L-1 2,4-D. As fases seguintes mostraram que é possível a diminuição ou retirada do regulador de crescimento, o que estimula o ciclo repetitivo e permite o desenvolvimento e conversão dos embriões somáticos. Foi observada a formação de embriões somáticos de forma indireta e direta. Houve conversão de embriões somáticos em plântulas e várias passaram para a aclimatização.

High-efficiency somatic embryogenesis and plant regeneration in California poppy, Eschscholzia californica Cham.

The development of a rapid protocol for high-efficiency somatic embryogenesis and plant regeneration from seed-derived embryogenic callus cultures of California poppy (Eschscholzia californica Cham.) is reported. The optimized procedure required less than 13 weeks from the initiation of seed cultures to the recovery of plantlets and involved the sequential transfer of cultures onto solid Murashige and Skoog basal medium containing three different combinations of growth regulators. All steps were performed at 25 °C. Friable primary callus was induced from seeds of E. californica cultured on medium supplemented with 1.0 mg l-1 2,4-dichlorophenoxyacetic acid. The primary callus was transferred to medium containing 1.0 mg l-1 1-naphthaleneacetic acid and 0.5 mg l-1 6-benzylaminopurine to establish embryogenic callus and promote somatic embryogenesis. Regenerated plantlets were recovered after the conversion of somatic embryos on medium containing 0.05 mg l-1 6-benzylaminopurine and showed normal development. Embryogenic callus was induced at a frequency of 85%, an average of 45 somatic embryos were produced per callus, 90% of the somatic embryos converted, and about 70% of the plantlets were recovered in soil. The growth rate of somatic embryo-derived shoots could be increased by gibberellic acid treatment, but the resulting plantlets were hyperhydritic.