Development and regeneration of somatic embryos from leaves-derived calli of Coffea liberica

Ardiyani F, Utami ESW, Purnobasuki H, Paramita SA. 2020. Development and regeneration of somatic embryos from leaves-derived calli of Coffea liberica. Biodiversitas 21: 5829-5834. Coffea liberica is an important and potentially commercial plant with a high economic value from the Coffea genus. Therefore, the availability of planting material is needed to increase productivity and ensure the sustainability of its farming. Somatic embryogenesis is a powerful propagation method used to produce clonal plants from limited genetic material. In the present research, we have shown that C. liberica could be successfully regenerated in vitro via somatic embryogenesis from leaves derived embryogenic callus. These calli were cultured on Murashige Skoog (MS) medium added with 1 mgL-1 BAP or in combination with 2.4 D (0.5, 1.0, 1.5 and 2 mgL-1) for embryo development induction. Furthermore, the medium containing only BAP was best for embryo development induction after culturing for 12 weeks, with the highest number of cotyledonary stage embryos (17.8%) and producing a total of embryo (20.2). Following cotyledonary stage embryo were cultured on new MS medium containing 0.5 mgL-1 BAP, 0.5 mgL-1 IAA, 0.5 mgL-1 NAA only, and 0.5 mgL-1 BAP in combination with 0.5 mgL-1 IAA or 0.5 mgL-1 NAA. Interestingly, the results showed that cotyledonary stage embryos were converted into complete plants at all treatment, but the MS medium containing 0.5 mgL-1 BAP was found to be the most effective in promoting regeneration with 2.6 leaves per-plantlet and height of 5.2 mm. Based morphological analysis confirm that the development of somatic embryo from leaves-derived calli of Coffea liberica started with the formation of embryo globular, heart, torpedo, cotyledonary stages, and finally conversion of cotyledonary embryo into complete plant.

Somatic Embryogenesis in Papaya (Carica papaya L. cv. TNAU Papaya CO.8)

An efficient indirect somatic embryogenesis protocol for Carica papaya var TNAU Papaya CO.8 was developed using immature zygotic embryos as an explant. Two growth regulators namely 2,4-D and picloram each at 1, 2, 3 mg/L were tested for callus induction and the highest callus induction frequency (83.33%) was observed in MS medium supplemented with 3 mg/L 2,4-D. However the rate of conversion into somatic embryos was highest (63.33%) on MS medium supplemented with 2 mg/L 2,4-D. Maturation of somatic embryos was studied by using MS medium with different concentrations of abscisic acid (ABA) and benzyl amino purine (BAP) along with glutamine (400 mg/L). The maturation of globular embryos was observed to be higher in the combination of ABA (1.5 mg/L), BAP (0.4 mg/L) along with glutamine (400 mg/L). Even though regeneration was observed from cotyledonary stage embryos in presence of different growth regulators like BAP, α-naphthalene acetic acid (NAA), phloridzin dehydrate kinetin and gibberellic acid, further growth was not observed due to abnormal regenerative structures. Regeneration of cotyledonary stage somatic embryos were highest (77.4%) in half strength MS medium without growth regulators. The well-developed plantlets with shoots and roots were subsequently transferred for hardening.

Early harvest increases post-harvest physiological quality of Araucaria angustifolia (Araucariaceae) seeds

<p><em>Araucaria angustifolia </em>is a native conifer from Brazil and an endangered species. Its seeds have a short period of viability, and this factor contributes to its vulnerability. This study aimed to evaluate physiological quality during the development and post-storage period of <em>A. angustifolia</em> seeds. Cones <em>A. angustifolia</em> were collected in the Curitibanos – Santa Catarina - Brazil in March, April, May, June, July and classified in cotyledonary, I, II, III and IV development stage according to the month of collection. Thereafter, seeds were stored in a refrigerator for 60 and 120 days and submitted to germination test in a germination chamber (25°C – photoperiod 12 h), moisture content (105 °C by 24 hours), tetrazolium test (0.1% by 1 hour) and vigor tests: electric conductivity (75 mL distilled water at 25 °C) and germination speed index, shoot, root length. During seed development, the moisture content decreased from the cotyledonary stage (66.54%) to stage IV (47.44%), and an increase in viability and vigor at the last stage, was observed. During the storage, the moisture content at the cotyledonary and I stage was stable. On the other hand, at stages II and III, stored seeds showed decrease after 120 days. Physiological quality at the cotyledonary stage increased germination to 86% and 93% after 60 and 120 days of storage, respectively. Unlike the stages II and III, that showed a decrease in seeds viability and vigor after storage. Electrical conductivity was higher for fresh seeds at the cotyledonary stage than for those stored for 60 days and 120 days. However, at other stages, released leachates content, after 120 days of storage, was higher with the advance of collection period. Germination speed index, shoot and root lengths showed seeds at cotyledonary and I stage had the highest values after storage period, unlike at stages II and III, where short root and shoot lengths were observed during storage. Thus, the maintenance of seed moisture content during storage was variable and dependent on the period of collection. Furthermore, the behavior of the physiological quality was different at the early and later stages. An early collection favored seeds physiological quality, and may be a strategy to increase the conservation of<em> A. angustifolia</em> seeds.</p>

Produção de sementes sintéticas de maracujazeiro silvestre com potencial ornamental

Passiflora cincinnata is a wild species of passion fruit with a wide geographical distribution. It has vigorous growth, climbing habit and very showy and fragrant flowers. The aim of the present investigation was to obtain synthetic seeds from encapsulated zygotic and somatic embryos of P. cincinnata, cultivated under different conditions. Precotyledonary and cotyledonary stage embryos were obtained from zygotic embryos cultivated on MS medium supplemented with 18.1 μM of 2,4-Acid-dichlorophenoxyacetic (2,4-D) and 4.5 μM of Benzyladenine (BA). Zygotic embryos and somatic embryos stages were encapsulated using sodium alginate (2.5% w v-1) and CaCl2.2H2O (1 mM) as complexing agent. The zygotic and somatic embryos were encapsulated in a matrix containing (I) sodium alginate, (II) sodium alginate + artificial endosperm and (III) sodium alginate + artificial endosperm supplemented with activated charcoal (0.15% w/v). Zygotic embryos encapsulated in the matrix (I), matrix (II) and matrix (III) and cultivated in flasks, germinated at rates of 79%, 76% and 86% respectively. The cotyledonary somatic embryos encapsulated in the 3 different matrices showed better germination rates when cultivated on cellulose plugs, with more than 50% of embryos converted into plants. Precotyledonary somatic embryos did not germinated regardless the matrix and cultivation. When cultivating the alginate beads ex vitro, both substrate Plantmax and Florialite showed low number of germinated embryos, and the best result (12.67%) were obtained using Florialite and embryos encapsulated in the matrix (I).

Two-dimensional gel electrophoretic protein profile analysis during seed development of Ocotea catharinensis: a recalcitrant seed species

The aim of the present work was to characterize changes in the protein profile throughout seed development in O. catharinensis, a recalcitrant species, by two-dimensional gel electrophoresis. Protein extraction was undertaken by using a thiourea/urea buffer, followed by a precipitation step with 10% TCA. Comparative analysis during seed development showed that a large number of proteins were exclusively detected in each developmental stage. The cotyledonary stage, which represents the transition phase between embryogenesis and the beginning of metabolism related to maturation, presents the highest number of stage-specific spots. Protein identification, through MS/MS analysis, resulted in the identification of proteins mainly related to oxidative metabolism and storage synthesis. These findings contribute to a better understanding of protein metabolism during seed development in recalcitrant seeds, besides providing information on established markers that could be useful in defining and improving somatic embryogenesis protocols, besides monitoring the development of somatic embryos in this species.

Storage protein dynamics in zygotic and somatic embryos of white fir

Composition and accumulation patterns of storage proteins in female gametophyte and embryos of the white fir (Abies concolor) were investigated during embryogenesis and germination of mature seeds using SDS-PAGE and immunological approach. Altogether 9 major and minor protein components with molecular masses of 14, 16, 22, 24, 27, 30, 35, 38, and 43 kDa were detected in female gametophytes and 9 protein bands in the embryos with the molecular sizes of 14, 16, 22, 24, 25, 27, 34, 38, and 43 kDa. The species seems to deviate in this respect from other representatives of Pinaceae. A conspicuous increase of storage protein synthesis was observed at the stage of fully cellularized female gametophytes and at the cotyledonary stage of embryo development. There exists a high degree of similarity between storage protein profiles of white fir zygotic and somatic embryos. Successive stages of somatic embryogenesis exhibited a high degree of similarity of storage proteins except for cotyledonary stage when a noticeable increase in storage protein synthesis was registered. Conversely, during germination of somatic embryos, an overwhelming majority of storage proteins was depleted.

Biomass, virus concentration, and symptomatology of cucurbits infected by mild and severe strains of Papaya ringspot virus

Pre-immunization with mild strains of Papaya ringspot virus - type W (PRWV-W) has allowed the mosaic disease to be controlled in different cucurbit species, with increases in marketable fruit yield. The objective of this study was to compare virus concentration, biomass and symptomatology of 'Caserta' zucchini squash, 'Menina Brasileira' long-neck squash and 'Crimson Sweet' watermelon plants infected by three mild strains and one severe strain of PRSV-W. Plants were inoculated at the cotyledonary stage, under greenhouse conditions, sampled at 7, 14, 21, 28 and 35 days after inoculation (DAI), and analyzed by PTA-ELISA. The severity of the symptoms was scored according to a scale from 1 to 5, and the fresh and dry biomass of the aerial part of the plants were evaluated at 40 DAI. Concentrations of the mild strains, based on absorbance values of the PTA-ELISA, were lower than the concentration of the severe strain for all species. The mild strains did not cause mosaic in infected plants of all species. Plants of zucchini squash and watermelon infected by the severe strain exhibited severe mosaic symptoms, but the same was not noticed for infected long-neck squash plants. Biomass values from zucchini squash and watermelon plants infected by the mild strains were 1.7 % to 12.4 % lower as compared to healthy plants. Biomass values of zucchini squash and watermelon plants infected by the severe strain presented greater reduction, varying from 29 % to 74 %. However, biomass values of long-neck squash plants infected by the mild and severe strains were similar for all treatments.

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.

Endogenous auxins and embryogenesis in Phaseolus coccineus

The occurrence and the dynamics of free and bound indole-3-acetic acid (IAA) in different parts of Phaseolus coccineus L. seeds were investigated at various developmental stages. Results show that free and bound IAA content in the single seed parts is quite different, and changes with different patterns during seed growth. The highest concentration of total IAA was found in early-heart stage embryos, while the total IAA concentration in the suspensor at early-heart stage is much lower than in the embryos, and remains almost constant in the later stages. Integuments have an intermediate content at the first two developmental stages, while showing the highest concentration of total IAA at the cotyledonary stage. Concerning the percentage of free IAA in relation to the total pool, we found that in the embryos free IAA accounts for 26–28% in the first two stages then increases up to 44% at the cotyledonary stage. In suspensors, the percentage of free IAA is much higher (90%) in all developmental stages. We discuss the idea that the different level of auxin between embryos and the suspensor might play an important role in the establishment of embryo polarity.

Compatibility Studies in Hydrangea

Improvement in cold-hardiness is needed in bigleaf hydrangea (Hydrangea macrophylla). Potential sources of cold-hardiness for enhancing the usefulness of this popular shrub include panicle hydrangea (H. paniculata), smooth hydrangea (H. arborescens), and oakleaf hydrangea (H. quercifolia). The objective of this study was to investigate the possibility of creating hybrids between these three species and H. macrophylla. In addition, self-compatibility of each of the species was evaluated to determine the need for emasculation when making controlled pollinations. Numerous cultivars of the species were utilized in both the self-pollination and interspecific hybridization studies. Number of flowers self-pollinated ranged from 1000 in H. arborescens to 3000 in H. quercifolia. Viable seeds were obtained from all species except H. arborescens. All five of H. quercifolia, 9 of the 18 H. paniculata, and 1 of the 8 H. macrophylla self progeny obtained survived. Emasculation of Hydrangea flowers to be used in controlled pollinations is therefore recommended. Seedlings were obtained from all interspecific crosses in which H. macrophylla was used as the maternal parent. All putative hybrid seedlings died either at the cotyledonary stage or while the first set of true leaves was expanding.