Embryo Maturation Medium Research Articles

Induction and maturation of somatic embryogenesis in mangaba tree a multipurpose Apocynaceae

This study reported the first results of in vitro somatic embryogenesis induction in Hancornia speciosa Gomes (mangaba tree, Apocynaceae). To induce callus, the TC accession leaf and nodal segments were cultivated in culture medium I (Murashige and Skoog; MS salts, putrescine, 45 µM 2,4-dichlorophenoxyacetic acid; 2,4-D and 22.19 µM 6-benzylaminopurine; BA). For the next stage (multiplication), the callus masses were sub-cultured into a culture medium II (MS salts, 22.62 µM 2,4-D and 22.19 µM BA). After 90 days, embryogenic calluses were inoculated to somatic embryo maturation medium culture III (MS salts, polyethylene glycol; PEG (0 and 2%) and BA (44.39 and 66.58 µM). Pro-embryos and somatic embryos in the maturation stage were assessed after 60 days. The rstatix package from R software with non-parametric tests (p < 0.05) were used to analysis data of experiments I and II. The Tukey’s test (p < 0.05) was performed on the data of experiment III. To induce and multiply embryogenic callus, 45 µM 2,4-D and 22.19 µM BA were efficient. More pro- and somatic embryo development were seen in the nodal explant cultured in the presence of 44.39 and 66.58 µM BA and 2% PEG. Putrescine does not promote the embryogenic masses of callus derived from leaf explants in the TC accession. Keywords: Hancornia speciosa Gomes, micropropagation, somatic embryo.

EVALUACIÓN DEL EFECTO DE LA PUTRESCINA EXÓGENA DURANTE LA MADURACIÓN Y GERMINACIÓN in vitro DE EMBRIONES SOMÁTICOS EN DOS GENOTIPOS DE AGAVE

<p><strong>Background.</strong> Agaves are an important economic source in industrial products, in the food and pharmaceutical industry, steroidal animal feed, ornamental plants and as a material for biofuels. Despite their economic importance, some species present problems in their conventional propagation, which makes their reproduction difficult and has caused some species to be in some risk category. Therefore, there is a need to develop efficient protocols for <em>in vitro</em> micropropagation via somatic embryogenesis. The positive effect of the application of exogenous putrescine on the maturation and germination of somatic embryos is known. <strong>Objective</strong>. To determine the effect of exogenous putrescine on the maturation and germination of somatic embryos of <em>A. cupreata</em> and <em>A. angustifolia</em>.<strong> Methodology</strong>. Two concentrations of putrescine (100 and 150 mg/L) and two controls, one positive and one negative, were evaluated in the somatic embryo maturation medium and later in the germination of somatic embryos. <strong>Results</strong>. Putrescine favors the maturation and germination of somatic embryos in both species. <strong>Implications</strong>. The results of this research contribute to a better understanding of the process of somatic embryogenesis and open the possibility of applying putrescine in the process of somatic embryogenesis at a commercial level. <strong>Conclusion</strong>. Exogenous putrescine in culture medium significantly improves the maturation and germination of somatic embryos in <em>Agave</em>.</p>

Effect of culture media, auxins and genotypes on plantlet regeneration from oil palm (Elaeis guineensis Jacq.) zygotic embryos through somatic embryogenesis

Aim: This study evaluated efficient culture media for the regeneration of elite material through somatic embryogenesis from oil palm zygotic embryos. Methodology: For callus induction, zygotic embryos of four elite genotypes (G1-264T, G2-238DX17P, G3-37DX17P and G4-237T) were cultured on three basal media (Y3, MS and N6) with different auxin 2 mg l-1 (Picloram, 2,4-D and Dicamba) combinations. Subculture was made every month for three passages. It evaluated various callus characters. The embryogenic calli from callus induction media were transferred to the embryo maturation medium and subcultured until the polyembryoids formed. For shoot and root formation, somatic embryo clumps were transferred into regeneration media. In-vitro plantlets with well-grown roots were hardened in pots for six weeks and assessed clonal fidelity using polymorphic SSR primers. Results: Among the treatments, calli from N6+2,4-D, Y3+2,4-D and N6+Picloram showed the highest embryogenic callus potential. G4-237T induced more embryogenic calli (32.982) among genotypes, which was on par with G1-264T (24.196). Embryogenic calli grown on N6 media with Dicamba showed the highest proliferation rate (1.141). After 60 days of culture on regeneration media, the highest number of plantlets per somatic embryogenic clump was obtained from G1-264T on N6 media supplemented with Dicamba. Interpretation: Culture media salt concentration showed a significant difference among media by causing perturbations of auxin flow during somatic embryogenesis affecting callus induction, proliferation and plantlet regeneration. This may be useful for standardizing the genotype-specific regeneration media in oil palm.

Plant regeneration via protoplast electrofusion in cassava

Protoplast electrofusion between callus protoplasts of cultivar TMS60444 and mesophyll protoplasts of cultivar SC8 was performed as an approach for the genetic improvement of cassava. The fusion products were subsequently cultured in protoplast culture medium (TM2G) with gradual dilution for approximately 1–2 months. Then the protoplast-derived compact calli were transferred to suspension culture medium (SH) for suspension culture. The cultured products developed successively into embryos, mature embryos, and shoots on somatic embryo emerging medium (MSN), embryo maturation medium (CMM), and shoot elongation medium (CEM), respectively. And the shoots were then rooted on Murashige and Skoog (1962) medium (MS). Sixty-six cell lines were obtained and 12 of them developed into plantlets. Based on assessment of ploidy level and chromosome counting, four of these plantlets were tetraploid and the remaining eight were diploid. Based on assessment of ploidy level and simple sequence repeat (SSR) analysis, nine tetraploid cell lines, one diploid variant plant line and nine variant cell lines were obtained. The diploid variant plant line and the nine variant cell lines all showed partial loss of genetic material compared to that of the parent TMS60444, based on SSR patterns. These results showed that some new germplasm of cassava were created. In this study, a protocol for protoplast electrofusion was developed and validated. Another important conclusion from this work is the confirmation of a viable protocol for the regeneration of plants from cassava protoplasts. Going forward, we hope to provide technical guidance for cassava tissue culture, and also provide some useful inspiration and reference for further genetic improvement of cassava.

Establishment of a somatic embryo regeneration system and expression analysis of somatic embryogenesis-related genes in Chinese chestnut (Castanea mollissima Blume)

Somatic embryogenesis is a reliable and important tool, and the relevant genes controlling this process act as vital roles through the whole development of somatic embryos. However, regeneration via somatic embryogenesis in Chinese chestnut has been impeded and its molecular mechanism is not known. Therefore, firstly we described a protocol for somatic embryo initiation, development, maturation and germination. Embryogenic calli were obtained in embryo initiation medium containing 1.8 μM 2,4-D and 1.1 μM 6-BA, and then were transferred to embryo development medium without any hormones for at least 4 weeks, until cotyledonary embryos appeared. Next, the somatic embryos were transferred to embryo maturation medium containing Gamborg’s B-5 Basal Salt Mixture with 0.5 μM NAA and 0.5 μM 6-BA for 3 weeks. Finally, these mature embryos were germinated in embryo germination medium consisting of WPM with 0.5 μM NAA and 0.5 μM 6-BA, resulting in shoot regeneration with a 2.1% conversion rate. Additionally, eight embryogenesis-related genes were identified, and the expression profiles of these genes during embryogenesis were analyzed via quantitative real-time RT-PCR (qRT-PCR). The CmSERK, CmLEC1, CmWUS and CmAGL15 genes exhibited high expression in the initial embryo stages, which inferred that these genes played key roles during the initiation of embryogenesis. Studies on embryogenesis-related genes will provide an insight for further elucidating molecular mechanism during somatic embryogenesis of Chinese chestnut. Furthermore, the successful establishment of a somatic embryo regeneration system for Chinese chestnut will lay a significant foundation for a stable genetic transformation system and genetic improvement.

A clonal propagation system for Atlantic white cedar (Chamaecyparis thyoides) via somatic embryogenesis without the use of plant growth regulators

Atlantic white cedar (AWC; Chamaecyparis thyoides), an aromatic evergreen conifer native to swamps and bogs along the Atlantic and Gulf coasts of the eastern United States was once an important species for timber production due to its durable wood. However, native populations have declined over the past two centuries. We established an in vitro propagation system for AWC via somatic embryogenesis (SE) without the use of plant growth regulators (PGRs). Whole megagametophytes with zygotic embryos from immature AWC cones were cultured on a modified half-strength embryo maturation (EM) medium with three different PGR treatments, including one devoid of PGRs. Both PGR treatment and cone collection date had significant effects on embryogenesis induction, with EM with no PGRs giving the highest embryogenesis induction, which ranged as high as 27%. We also conducted experiments to determine the effects of activated carbon (AC) and abscisic acid (ABA) in the maturation medium on production of mature somatic embryos. AC significantly affected this variable, with 2 g l−1 producing more embryos than 0 g l−1. Application of exogenous ABA not only failed to improve production of mature somatic embryos, the highest level tested (200 µM), apparently lowered production of mature embryos compared to the 0 ABA control. The highest numbers of mature somatic embryos per ml of plated embryogenic suspension (32–37) were produced on medium with 2 g l−1 AC and levels of ABA at 100 µM or lower. The SE system described here has the potential to contribute the restoration of Atlantic white cedar to its native habitat.

Efficient Plant Regeneration of Selected Kenyan Sweetpotato (Ipomoea batatas (L.) Lam.) Cultivars through Somatic Embryogenesis

Sweetpotato is an important food crop in the world as well as in Kenya. Various fungal, and viral diseases are major constraints in its production and are currently threatening the sweetpotato production in sub-Saharan Africa. Genetic engineering offers significant potential for the crop’s genetic improvement. However, this is limited by the low efi¬Âciency and strong genotype dependency in tissue culture. This study aimed to establish an efficient somatic embryogenesis and plant regeneration system using shoot apical meristem explants of sweetpotato. Three sweetpotato cultivars that are widely grown in Kenya; KSP36, Kemb36 and Mweu mutheke along with an exotic model cultivar Jewel were evaluated. The maximum somatic embryogenic induction, at 96.72%, was obtained from explants cultured on Linsmaier and Skoog salts and vitamins medium supplemented with 0.5 mg/l dichlorophenoxyacetic acid and 0.2 mg/l zeatin riboside. The highest number of shoot induction (33) was observed after transfer of embryonic callus to embryo maturation medium supplemented with 2 mg/l abscisic acid. Significant differences were observed between cultivars for somatic embryogenesis and plant regeneration. Jewel showed the best response, while Mweu mutheke was the least responsive under the culture conditions tested in this study. Regenerated plants were successfully rooted and grown to maturity after hardening in soil in the greenhouse. Such a robust, successful and efi¬Âcient system possesses the potential to become an important tool for crop improvement and functional studies of genes in sweetpotato.

Efficient plant regeneration of watermelon (Citrullus lanatus Thunb.) via somatic embryogenesis and assessment of genetic fidelity using ISSR markers

A simple and effective somatic embryogenic system was established for watermelon (Citrullus lanatus) cv. ‘Arka Manik’. Embryogenic callus was obtained from leaf explants of 20-d-old in vitro-grown seedlings cultured on embryogenic callus induction medium. The highest frequency of embryogenic callus induction (96.8%) occurred on Murashige and Skoog (MS) medium supplemented with 2.44 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.27 μM thidiazuron (TDZ). Transfer of embryogenic calluses with proembryogenic masses to embryo maturation medium led to the asynchronous development of somatic embryos (SEs), which progressed from the globular stage to the cotyledonary stage. The maximum number of SEs/explant (16.1 ± 0.24) was obtained on MS medium supplemented with 2.44 μM 2,4-D, 2.27 μM TDZ, and 30 g L−1 sucrose. Plantlet conversion from cotyledonary-stage SEs was tested on different strengths of MS medium (quarter-, half-, and full-strength) lacking plant growth regulators. The highest frequencies of germination (91.5%) and survivability (82.1%) of plantlets were achieved on full-strength MS medium. Transverse sections of embryogenic callus revealed SE development from callus cells near the epidermis. Secondary SEs occasionally formed from globular-shaped primary embryos. Genetic fidelity of mother plants and ex vitro plants was confirmed by inter-simple sequence repeat (ISSR) markers. The present study is the first report on the use of molecular markers in in vitro culture of watermelon. The developed protocol facilitates rapid production of true-to-type watermelon plants by somatic embryogenesis and thus could serve to generate effective target material for genetic transformation protocols.

Effect of Gelling Agent and Selective Sub-culturing on Hyperhydricity in Anther-Derived Coconut Embryos

Experiments were conducted to compare the liquid medium with the media solidified with agar or phytagel. Selective subculturing and use of the embryo maturation medium supplemented with higher concentration of phytagel (0.5%; w/v) were also tested for reducing the vitrified embryos. Modified Eeuwens Y3 medium was used as the basal medium. By culturing the anthers on the medium solidified with phytagel (0.25%; w/v), direct embryo formation (86.7%) and embryo conversion (21.5%) were significantly increased. Plant regeneration efficiency of anther derived embryos or calluses developed in the liquid culture medium was extremely low (2.4%). Vitrification was further reduced by incorporating 0.5% (w/v) phytagel into the embryo maturation medium. Highest plant regeneration efficiency was obtained by exposing the embryos to 0.5% (w/v) phytagel for 21 days, which reduced vitrification by 42%. Furthermore, selective subculturing of the embryos was effective for reducing vitrification.

Agrobacterium-mediated transformation using embryogenic calli in Satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa wase

Agrobacterium-mediated transformation in Satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa wase was achieved with reasonable transformation efficiency of about 22%, which was the percentage of transgenic plantlets confirmed by genomic PCR (37 plantlets/168 hygromycin-resistant calli). Embryogenic calli of Miyagawa wase were infected with Agrobacterium tumefaciens strain EHA105 harboring binary vector pCAMBIA1300 that contained miraculin gene (a taste-modifying protein) and hygromycin as a selection marker. After 5 days of co-culture in the medium containing 100 μM acetosyringone, calli were transferred to the liquid half embryogenic cell culture medium (half concentration of Murashige and Tucker’s (MT) medium modified with the addition of 500 mg·L− malt extract, 50 g·L− sucrose and 1.55 g·L− glutamine) with 15 mg·L− hygromycin and 250 mg·L− cefotaxime, and were cultured for two weeks. Then, the calli were grown on the solid selection medium with 20 mg·L− hygromycin for four weeks and 25 mg·L− hygromycin for another four weeks. The resistant embryos were selected and transferred to the embryo maturation medium. After 3 weeks of culture, the heart shaped embryos were transferred to the MT medium containing 1.0 mg ·L− GA3, 20.0 mL·L− coconut water, 0.02 mg·L− NAA and 0.0146 mg·L− coumarin for embryo germination. Finally the germinated embryos were cultured on MT medium containing 3.0% sucrose and 0.8% agar for growing to the normal plant. Stable integration of the transgene in the plant genome was confirmed by PCR and Southern blot analysis.

Efficient and rapid plant regeneration of oil palm zygotic embryos cv. ‘Tenera’ through somatic embryogenesis

An efficient and rapid plant regeneration system through somatic embryogenesis was developed using 13-week-old zygotic embryos of oil palm (Elaeis guineensis Jacq.) cv. ‘Tenera’. Zygotic embryos were cultured on MS and N6 media supplemented with 2.0 mg L−1 picloram, 2,4-D and dicamba. The highest embryogenic callus formation (32%) was observed on N6 medium with 2,4-D after 3 month culture on callus induction medium. Somatic embryos were continuously formed from nodular calli on embryo maturation medium [N6 + 0.1 mg L−1 2,4-D, 0.16 g L−1 putrescine, 0.5 g L−1 casein amino acids and 2.0 g L−1 activated charcoal(AC)] for 3–5 months. Histological analysis confirmed that embryo development occurred via somatic embryogenesis. For plant regeneration, modified N6 medium (MN6) with AC (0.5 g L−1) without growth regulators, induced both shoot and root formation simultaneously with the highest regeneration rate of 56%. This combined shoot and root induction protocol shortened the culture time to 9–12 months. Furthermore, after acclimatization, more than 85% of transferred plants from our protocol developed successfully in the soil.

Changes in H2O2 content and antioxidant enzyme gene expression during the somatic embryogenesis of Larix leptolepis

Hydrogen peroxide (H2O2) content and transcript levels of genes encoding superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) antioxidant enzymes were investigated during different stages of somatic embryogenesis in Larix leptolepis. H2O2 content was lowest on day 0 when embryogenic callus was incubated on Murashige and Skoog (MS) medium supplemented with polyethylene glycol (PEG). This content began to increase when callus was transferred to the same medium but containing abscisic acid (ABA), and reached a peak at day 3 following incubation. The level of H2O2 dropped from day 7 to day 10, peaked at day 21, then dropped again at day 24 and day 35, but increased when somatic embryos reached maturity at day 45. Transcript levels of SOD, CAT, and APX were lowest when somatic embryos were cultured on callus induction medium without ABA. When calli were transferred onto somatic embryo maturation medium, expression patterns of SOD, CAT, and APX varied, and transcript levels at all stages were higher than those at d 0. SOD expression was highest at day 3. Whereas, CAT expression levels were low during early stages of somatic embryogenesis, but increased at day 21, declined at days 24 and 35, and then began to increase again at day 45. APX gene expression patterns were highest at days 3, 21, and 45. These results suggested that ABA was essential for promoting somatic embryogenesis of L. leptolepis. Moreover, ABA induced production of H2O2 and other active oxygen species (AOS), and mediated CAT, SOD, and APX gene expression in somatic embryogenesis of L. leptolepis.

Production of plants resistant to Alternaria carthami via organogenesis and somatic embryogenesis of safflower cv. NARI-6 treated with fungal culture filtrates

The present study describes a system for efficient plant regeneration via organogenesis and somatic embryogenesis of safflower (Carthamustinctorius L.) cv. NARI-6 in fungal culture filtrates (FCF)-treated cultures. FCF was prepared by culturing Alternaria carthami fungal mycelia in selection medium for host-specific toxin production. Cotyledon explants cultured on callus induction medium with different levels of FCF (10–50%) produced embryogenic callus. In organogenesis, 42.2% microshoots formed directly from embryogenic callus tissues in plant regeneration medium with 40% FCF. Isolated embryogenic callus cultured on embryo induction medium containing 40% FCF induced 50.2% somatic embryogenesis. Embryo germination percentage was decreased from 64.5 to 28 in embryo maturation medium containing 40% FCF. However, nine plantlets from organogenesis and 24 plantlets from somatic embryogenesis were selected as FCF-tolerant. Alternaria carthami fungal spores (5 × 105 spores/ml) sprayed on the leaves of FCF-tolerant plants showed enhanced survival rate over control plants, which plants were more susceptible to fungal attack. The number of leaf spot lesions per leaf was decreased from 3.4 to 0.9 and their lesion length was also reduced from 2.9 to 0.7 mm in organogenic derived FCF-tolerant plants over control. In somatic embryo derived FCF-tolerant plants, the number of lesions was decreased from 3.1 to 0.4 and the lesion size was also reduced to 2.7–0.5 mm when compared to the control. This study also examined antioxidant enzyme activity in FCF-tolerant plants. Catalase (CAT) activity was slightly decreased whereas peroxidase (POD) activity was increased to a maximum of 42% (0.19 μmol min−1 mg−1 protein) from organogenesis and 47% (0.23 μmol min−1 mg−1 protein) from embryogenesis in FCF-tolerant plants. Superoxide dismutase (SOD) activity was also increased to 17% (149 U mg−1 protein) and 19.5% (145 U mg−1 protein) in FCF-tolerant plants derived from organogenesis and somatic embryogenesis when compared with control plants.

EMBRIOGENESIS SOMATIK ANGGREK BULAN Phalaenopsis amabilis (L.) Bl: STRUKTUR DAN POLA PERKEMBANGAN

Research of the structure and development pattern of somatic embryos from callus of leaf explants moon orchid Phalaenopsis amabilis (L) Bl had been done. One year old of plantlets were used as explants sources. Basal leaf of these explants were cultured in Somatic Embryo Induction Medium (SEIM) e.i.: NP(New Phalaenopsis) medium added with 2 mg/L NAA, 1 mg/L BA, 10 g/L sucrose, and 2 g/L gellan gum. Then somatic embryos were transferred to EMM (Embryo Maturation Medium) e.i. NP medium added with 1 mg/L NAA, 1 mg/L BA, 10 g/L sucrose, and 2 g/L gellan gum. Finally, mature somatic embryo were transferred to NP medium without plant growth regulator as Embryo Germination Medium (EGM). The origin of somatic embryos initially from single cell at the pheriphery of embryogenic callus. These cells then devided in mitotic repeatedly formed globular proembryo, elongation embryo, and completed embryo. The structure and development pattern of somatic embryos as the same as with zygotic embryo.

A simple and rapid Agrobacterium -mediated transformation protocol for cotton ( Gossypium hirsutum L.): Embryogenic calli as a source to generate large numbers of transgenic plants

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3-6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.

Somatic embryogenesis and plant regeneration in Gymnema sylvestre

Somatic embryogenesis and whole plant regeneration were achieved in callus cultures derived from hypocotyl, cotyledon and leaf explants excised from seedlings of Gymnema sylvestre. Embryogenic callus was induced on Murashige and Skoog (MS) medium containing 2,4-D (0.5–5.0 μM) +BA (0.5–2.0 μM) and 2% (w/v) sucrose in 6–8 weeks of culture. Globular/heart stage embryos developed on induction medium. These embryos produced torpedo and cotyledon stage embryos upon sub-culturing on embryo maturation medium EM8 (medium containing MS salts, B5 vitamins, 0.5 μM BA and 2% sucrose). Embryo germination and plantlet formation was achieved by sub-culturing mature embryos on fresh EM8 medium. The plantlets were acclimatized in the greenhouse.

Improvement of Direct Somatic Embryogenesis in Rice by Selecting the Optimal Developmental Stage of Explant and Applying Desiccation Treatment

SummaryBy selecting the optimal developmental stage of zygotic embryos used as expiants and applying desiccation treatment, we improved direct somatic embryogenesis in rice scutellum from two cultivars, Nipponbare and Sasanishiki. Zygotic embryos isolated 14-17, 21, 28-30 and 35-40 d after anthesis (DAA) from Nipponbare and 14-17, 18-21, 28-30 and 40-42 DAA from Sasanishiki were cultured on the embryo induction medium (EIM). Then they were transferred to embryo maturation medium (EMM) and germinated on the embryo germination medium (EGM). Only immature zygotic embryo isolated 14-17 DAA from Nipponbare and Sasanishiki could develop somatic embryos that germinated. Explants from embryos at other developmental stages could develop somatic embryos only until the elongating or scutellar stage. They enlarged and formed callus without further development. The EIM and EMM consisted of N6 macronutrients, B5 micronutrients, and B5 vitamins, supplemented with 0.1 g L-1 casein hydrolysate, 1.5 g L-1 proline and 1 g L-1 MES buffer. EGM consisted of MS macro- and micronutrients and MS vitamins without organic supplement. In addition, 2 mg L-1 2,4-D was added to EIM, 1 mg L-1 2,4-D to EMM and 0.01 mg L-1 zeatin to EGM. Developmental processes of somatic embryos derived from the expiants were observed by scanning electron microscopy. Desiccation treatment of maturing somatic embryo was proved to produce fully mature somatic embryos capable of germinating vigorously.

Genotype and auxin influence direct somatic embryogenesis from protoplasts derived from embryogenic cell suspensions of Asparagus officinalis L.

Embryogenic callus from four asparagus genotypes, Jersey Giant No. 8, MD10, Rutgers 22, and 86SOM1 was simultaneously initiated from spear explants on semisolid LS medium containing 5 μM 2,4-D or 50 μM NAA. Calluses were used to initiate cell suspensions in liquid LS medium of the same composition. The eight sets of cell suspensions were used as protoplast donors at both 2 and 5 months of age. Protoplasts were immobilized at 10 5/ml density in MS medium with 0.6% agarose and overlaid with liquid KM medium; both containing the same type and concentration of auxin used for the corresponding donor cells or with plant growth regulator-free (PGR-free) medium. There was a significant interaction between genotype, suspension auxin, and inclusion or exclusion of PGRs in the protoplast culture media on plating efficiency, and colony and somatic embryo formation. Plating efficiencies at 14 days ranged from 0–40%. Globular somatic embryos developed directly from protoplasts in 10–14 days and bipolar embryos could be transferred in 3–4 weeks to embryo maturation medium (EM medium) composed of LS medium with 2% sucrose and 1% Phytagel. Conversion to plants occurred as rapidly as 1–2 weeks after transfer to EM medium or 5–6 weeks after initial protoplast culture. Although all four genotypes regenerated plants, Rutgers 22 had the highest conversion frequency at 42%. Most plants recovered from the 2,4-D-derived protoplasts were karyotypically aberrant while a higher frequency of normal plants were obtained from the NAA-derived protoplast cultures.

886 PB 460 SOMATIC EMBRYOGENESIS FROM IMMATURE COTYLEDONS OF TEXAS BLUEBONNET

This study was initiated to test the embryogenic potential of immature cotyledons (3-5 mm long) of Texas bluebonnet (Lupinus texensis). The embryo initiation medium consisted of B5 salts and vitamins with 3% sucrose and 22.6 μM 2,4-D alone or in combination with 1-15 μM of various cytokinins. Within 15-20 days, globular embryos were formed on the distal end of the cotyledons. Eventually the entire cotyledon surface was covered by embryo-like structures. Addition of cytokinins to the medium did not increase the percentage of cultures which formed embryos. In fact, addition of thidiazuron severely inhibited embryogenesis. Following transfer to an embryo maturation medium (MS medium with 0.38 μM abscisic acid alone or in combination with benzyladenine or zeatin) for 10-14 days, the embryos were placed in MS medium supplemented with GA (2.9 μM) or glutamine (200 mg/liter) with or without activated charcoal (0.5%) for embryo germination and plantlet development. Most of the embryos exhibited precocious germination and well-developed roots but failed to produce normal shoots. Therefore, additional work is needed to improve embryo conversion frequency.

Development of haploid asparagus embryos from liquid cultures of anther-derived calli is enhanced by ancymidol

The effect of ancymidol concentration on the development of haploid asparagus embryos was determined. Liquid cultures from anther-derived calli were grown for three weeks in MS medium plus 1.0 mg l(-1) 2,4-D, 0.1 mg l(-1) NAA, 0.2 mg l(-1) kinetin, 800 mg l(-1) glutamine, 500 mg l(-1) casein hydrolysate, 2% sucrose and 0.0-1.0 mg l(-1) ancymidol. Cell clumps (224-500 μm) were plated on solid embryo maturation medium (MS medium plus 3% sucrose, 0.1 mg l(-1) NAA, 0.5 mg l(-1) kinetin and 0.0-1.0 mg l(-1) ancymidol) and grown for eight weeks. Ancymidol enhanced embryo maturation and germination and was more critical in the solid than liquid medium. Total embryo number did not vary among most treatments. The best response was observed when ancymidol concentrations were 0.1 and 0.5 mg l(-1) in the liquid and solid media, respectively; two-thirds of the embryos produced were bipolar and 35% of bipolar embryos germinated. Seven to 82% of plants recovered from different ancymidol treatments were haploid; the others were diploid, triploid or chimeric for ploidy level.