Immature Zygotic Embryos Research Articles

Embriogénesis y organogénesis de aguacate criollo mexicano

La variedad Drymifolia de aguacate (Persea americana Mill.) es usada generalmente como portainjerto por lo que es importante implementar las técnicas de embriogénesis somática y la organogénesis in vitro para su mejora genética, conservación y propagación clonal. En este trabajo se evaluaron embriogénesis somática directa, indirecta y organogénesis de la variedad Drymifolia. La germinación de los embriones somáticos maduros se indujo con 0.5 mg L-1 de 6-N-bencil amino purina (BAP) y 1 mg L-1 de ácido giberélico (GA3). De las seis accesiones evaluadas, Celaya 79, Comonfort 53, San Miguel, BG24, BG181 y Zutano, solo San Miguel respondió al proceso de embriogénesis. En la embriogénesis directa la eficiencia más alta de regeneración se obtuvo con 0.2 mg L-1 de picloram (46%) y 10 mg L-1 de ANA (40%). En la embriogénesis indirecta la accesión San Miguel formó callos con 0.2 mg L-1 de picloram y tuvo una eficiencia de regeneración de 45% conservando su potencial de regeneración hasta por seis meses. Con respecto a la organogénesis, se cultivaron embriones cigóticos inmaduros decapitados en medio con reguladores de crecimiento o sin ellos y las seis accesiones respondieron positivamente a ambas condiciones. La accesión Comonfort 53 tuvo mayor eficiencia de regeneración (54%) con reguladores de crecimiento. Este estudio realizado del 2016 a 2018, proporciona un enfoque nuevo y prometedor para la regeneración y multiplicación de plantas de P. americana var. Drymifolia a través de embriones cigóticos.

Somatic embryogenesis in slash pine (Pinus elliottii Engelm): improving initiation of embryogenic tissues and maturation of somatic embryos

Slash pine (Pinus elliottii Engelm.) has strong adaptability, early growth and high turpentine yield, and it is widely planted in southern China. Breeding of disease-resistant varieties is an effective way to prevent and control development of pine needle brown spot on slash pine in China. Plant regeneration by somatic embryogenesis has been achieved for slash pine, but large-scale production of somatic embryos remains restricted by several factors. We tested different embryogenesis conditions from time of initiation to maturation and studied genetic stability at simple sequence repeat (SSR) loci of regenerated plants in slash pine. Immature zygotic embryos of four open-pollinated mother trees were used for initiation of somatic embryogenesis. Seed sources (families) had a significant impact on initiation of embryonal suspensor mass (ESM, p < 0.05); however, the addition of abscisic acid ABA (1–2 mg/L) and phytosulfokine PSK (0.5 mg/L) could effectively improve initiation rate by up to 36%. Seed family 27 was the most favorable female parent for ESM initiation. The production (number of somatic embryos) was significantly increased by adding ABA (5 mg/L) during suspension culture before transferring to maturation media (p < 0.05). We tested the genetic stability of 14 regenerated plants at SSR loci, and 88.9% plants with normal phenotypes were found not to have genetic variation. We clarified the somatic embryogenesis conditions suitable for slash pine and established somatic embryo maturation technology. We clarified the somatic embryogenesis conditions suitable for slash pine and established somatic embryo maturation technology by studying several related influencing factors.

Indirect somatic embryogenesis and regeneration of Fraxinus mandshurica plants via callus tissue

Somatic embryogenesis of Fraxinus mandshurica has the problems of low somatic embryo (SE) yield, unsynchronized SE development, and a high percentage of deformed SEs. We aimed to improve F. mandshurica SE production by synchronizing SE development, improving SE quality, and inducing root formation to obtain complete regenerated plants. Cotyledons of immature zygotic embryos of F. mandshurica were induced to form callus and then SEs. The SE induction percentage from explants differed among 32 mother trees, and the one with the highest SE induction percentage (29.8%) was used for further experiments. The highest callus induction percentage was 94.2% on ½-strength Murashige and Skoog medium (MS½) supplemented with 0.15 mg·L−1 naphthalene acetic acid. The highest callus proliferation coefficient (240.5) was obtained on McCown’s Woody Plant Medium containing 0.1 mg·L−1 6-benzyl adenine and 0.15 mg·L−1 2, 4-dichlorophenoxyacetic acid. The highest number of SEs (1020.5 g−1 fresh weight) was obtained on MS½ medium supplemented with 1 mg·L−1 6-benzyladenine. The highest number of cotyledon embryos (397/g fresh weight) was obtained by incubating materials on medium containing 1 mg·L−1 abscisic acid and then applying a drying treatment. The cotyledon embryos were milky white, uniformly sized (average length 4.7 mm), and 80% of them were normal. The SE rooting percentage on ½MS medium containing 0.01 mg·L−1 NAA was 37.5%. Overall, the germination percentage of SEs was 26.4%, and complete regenerated plants were obtained after transplanting and acclimation. These results provide more possibilities for the preservation and breeding of F. mandshurica.

Regulation of cell reprogramming by auxin during somatic embryogenesis.

How somatic cells develop into a whole plant is a central question in plant developmental biology. This powerful ability of plant cells is recognized as their totipotency. Somatic embryogenesis is an excellent example and a good research system for studying plant cell totipotency. However, very little is known about the molecular basis of cell reprogramming from somatic cells to totipotent cells in this process. During somatic embryogenesis from immature zygotic embryos in Arabidopsis, exogenous auxin treatment is required for embryonic callus formation, but removal of exogenous auxin inducing endogenous auxin biosynthesis is essential for somatic embryo (SE) induction. Ectopic expression of specific transcription factor genes, such as "LAFL" and BABY BOOM (BBM), can induce SEs without exogenous growth regulators. Somatic embryogenesis can also be triggered by stress, as well as by disruption of chromatin remodeling, including PRC2-mediated histone methylation, histone deacetylation, and PKL-related chromatin remodeling. It is evident that embryonic identity genes are required and endogenous auxin plays a central role for cell reprogramming during the induction of SEs. Thus, we focus on reviewing the regulation of cell reprogramming for somatic embryogenesis by auxin.

Adventitious shoot-based propagation of Franklinia alatamaha for commercial horticulture and restoration

Franklinia is a monotypic genus of the family Theaceae that is now extinct in the wild. F. alatamaha Bartram ex Marshall has been maintained in cultivation for nearly 250-y because of the ornamental appeal of its flowers and foliage. The goal of this research was to develop an in vitro propagation system to increase the availability of this rare tree for commercial horticulture and restoration. Immature zygotic embryos were cultured on semi-solid Woody Plant Medium containing 34 different plant growth regulator treatments. Shoot organogenesis was induced from dark-grown explants with indole-3-butyric acid in combination with 6-benzylaminopurine. Shoot organogenesis was also obtained with thidiazuron, N-(2-Chloro-4-pyridyl)-N′-phenylurea, and indole-3-butyric acid in combination with meta-Topolin, although shoot development was abnormal. Adventitious buds were produced on the epidermal surfaces of immature zygotic embryos while exposed to the above plant growth regulator treatments. Once the adventitious buds elongated into shoots, these adventitious shoots were further multiplied by axillary shoot production. Elongated shoots from axillary shoot production were rooted on Woody Plant Medium supplemented with 0.25 g L−1 activated charcoal, but no plant growth regulators. The resulting plantlets were transferred to soil and acclimatized to the greenhouse and outdoor environments. Tissue culture-derived trees flowered within one and a half years in the greenhouse under an extended photoperiod.

Pinus canariensis plant regeneration through somatic embryogenesis

Aim of the study: To develop an efficient method to regenerate plants through somatic embryogenesis of an ecologically relevant tree species such as Pinus canariensis.Area of study: The study was conducted in the research laboratories of Neiker-Tecnalia (Arkaute, Spain).Material and methods: Green cones of Pinus canariensis from two collection dates were processed and the resulting immature zygotic embryos were cultured on three basal media. The initiated embryogenic tissues were proliferated testing two subculture frequencies, and the obtained embryogenic cell lines were subjected to maturation. Germination of the produced somatic embryos was conducted and acclimatization was carried out in a greenhouse under controlled conditions.Main results: Actively proliferating embryogenic cell lines were obtained and well-formed somatic embryos that successfully germinated were acclimatized in the greenhouse showing a proper growth.Research highlights: This is the first report on Pinus canariensis somatic embryogenesis, opening the way for a powerful biotechnological tool for both research purposes and massive vegetative propagation of this species.Keywords: acclimatization; Canary Island pine; micropropagation; embryogenic tissue; somatic embryo.Abbreviations used: embryogenic tissue (ET); established cell line (ECL); somatic embryogenesis (SE); somatic embryos (Se’s).

Somatic embryogenesis and cryopreservation of Stewartia species

Stewartia is a genus of flowering shrubs and trees in the family Theaceae. Stewartia species are native to eastern Asia, with the exception of S. ovata and S. malacodendron, which are indigenous to southeastern North America. Despite having outstanding ornamental value and unique horticultural features, Stewartias are not readily available for landscaping in the horticultural trade due to difficulty with mass propagation. In addition to commercial propagation, there is a need to develop propagation techniques for the conservation of the North American species, which are rare (S. ovata) or endangered (S. malacodendron). This research examined somatic embryogenesis as a means to mass propagate Asian and North American Stewartia species and to provide material for cryopreservation of valuable Stewartia germplasm. By testing a variety of plant growth regulators (PGRs), somatic embryogenesis was induced from immature zygotic embryo explants of seven Stewartia species—five Asian and two North American. Additionally, embryogenic cultures were obtained from three North American cultivars, S. malacodendron ‘Delmarva’, S. ovata ‘Red Rose’, and ‘Royal Purple’. For two Stewartia species, S. ovata and S. sinensis, somatic embryogenesis was induced from immature endosperm. Picloram (0.05 or 0.1 mg/L) and 2,4-dichlorophenoxyacetic (2 or 4 mg/L) were the most effective PGRs for inducing somatic embryogenesis of North American and Asian Stewartia species, respectively. All seven Stewartia species examined produced somatic embryos that converted into plants that could be transferred to the greenhouse. Embryogenic tissue from five Stewartia species were successfully put into cryo-storage and recovered, providing a means of long-term germplasm storage. This is the first report of somatic embryogenesis in the genus Stewartia. This is the first report of somatic embryogenesis from immature zygotic embryos in the genus Stewartia and it was accomplished with seven different species.

Somatic embryogenesis and plant regeneration in Betula platyphalla

Betula platyphylla is a native tree species in northern China that has high economic and medicinal value. We developed an efficient protocol for the induction of somatic embryogenesis in B. platyphalla from immature zygotic embryos and assessed the effects of explant type, genotype, and plant growth regulators (PGRs) on embryogenic callus induction. Among the various explants evaluated, embryogenic callus was only produced from mature and immature zygotic embryos on medium with added 2,4-dichlorophenoxyacetic acid (2,4-D). Supplementation of 2,4-D-containing medium with cytokinins increased the frequency of embryogenic callus induction. On the 20 days after pollination, immature zygotic embryos that had been collected in mid-May yielded embryogenic tissue at the highest frequency (16.8%) when cultured on half-strength MS medium supplemented with 2.0 mg L−1 2,4-D and 0.2 mg L−1 6-benzylaminopurine (6-BA). The process of proliferation of embryogenic callus, somatic embryo formation, and subsequent plantlet conversion occurred under optimal culture conditions. When regenerated plants were transplanted to soil, 95% of them developed normally and grew vigorously. This somatic embryogenesis system required 3–4 months for the regeneration of B. platyphalla plantlets from immature zygotic embryos.

Differential Influence of Growth Regulators during Somatic Embryogenesis of Gynodioecious Papaya Varieties‘CO.7’ and ‘Red Lady

The study involved two auxins viz., 2,4-D (2,4-Diclorophenoxyacetic acid) and picloram at three different concentrations (1,2, 3 mg/L) in full strength MS media to study their comparative influence on induction of somatic embryogenesis from immature zygotic embryos of two gynodioecious varieties of papaya ‘CO.7’ and ‘Red Lady’. In papaya cultivar ‘CO.7’, 2,4-D at 2 mg/L gave the highest callus induction frequency of 90.93%, whereas comparatively higher concentration of 3 mg/L 2,4-D was found suitable for ‘Red Lady’ (87.26%). Although there was profuse callus formation, 2 mg/L 2,4-D recorded comparatively higher frequency of embryogenic calli in ‘Red Lady’ (51.67%) when compared to ‘CO.7’ (30.00%). Somatic embryo maturation was achieved upon transfer of embryogenic calli exhibiting globular stage embryos on to maturation medium (MS medium + ABA (Abscisic acid) and BAP (Benzyl amino purine) in different concentrations + glutamine 400 mg/L). In the maturation medium, the combination of 1.5 mg/L ABA and 0.4 mg/L BAP registered better conversion of the globular embryo to cotyledonary embryos than other levels. The frequency of somatic embryo germination was higher in ‘Red Lady’ (50.00%) as compared to ‘CO.7’ (31.67%) on half-strength MS medium devoid of growth regulators.

The effect of auxins in inducing organogenesis or somatic embryogenesis in mature sunflower zygotic embryo derived apex

Induction of shoots or of somatic embryos is the key step for gaining the morphogenetic potential in sunflower (Helianthus annuus L.), species known as recalcitrant to in vitro regeneration. In the immature zygotic embryo derived tissues or in other juvenile tissues resulted from seedlings, the acquisition of the competence for regeneration can be achieved directly by cytokinin treatment or by preconditioning the explants on cytokinin containing medium. In this paper is presented a new type of explant for sunflower in vitro culture, consisting of the apex with primordial leaves, resulted from ungerminated mature zygotic embryo, in which a specific morphogenetic response was triggered by the exogenously applied auxins. Among the auxins tested, indole-3-acetic acid, indole-3-butyric acid and 1-naphthaleneacetic acid are inducers of an organogenetic response, apical/axillary shoots and adventitious buds being regenerated while 2,4-dichlorophenoxyacetic acid, 3,6-dichloro-2-methoxybenzoic acid and 4-amino-3,5,6-trichloropicolinic acid led to somatic embryo formation. Among the auxins tested only 4-amino-3,5,6-trichloropicolinic acid sustains the embryos development up to mature stage. A high amount of sucrose (120 g L-1) supplied during the auxin treatment promotes the maturation of the embryos directly on the induction medium for all tested auxins with embryogenic effect. These findings show that regardless of the type of morphogenetic response aimed in sunflower meristematic tissues resulted from mature embryos, the presence of auxins is mandatory.

Somatic Embryogenesis and Plantlet Regeneration in the Carica papaya L. cv. Eksotika.

A highly efficient protocol for regeneration of Carica papaya L. cv. Eksotika somatic embryos from immature zygotic embryos was developed. This study was designed to overcome the obstacles in regeneration of somatic embryos from immature zygotic embryos of “Eksotika”, especially problems associated with formation of better root quality and callus formation at the base of somatic embryos. Somatic embryos were generated by incubation of immature zygotic embryos in half-strength salt Murashige and Skoog (MS) medium with full-strength vitamins supplemented with 7.5 mg L−1 2,4-D, 100 mg L−1 L-glutamine, 50 mg L−1 myo-inositol, 45 mg L−1 adenine sulphate, 0.33% gelrite, and 6% sucrose, followed by transfer to maturation medium consisting of ½ MS medium supplemented with 5 mg L−1 phloroglucinol, 100 mg L−1 L-glutamine, 100 mg L−1 myo-inositol, 68 mg L−1 adenine sulphate, 0.38% gelrite, and 3% sucrose. After that, well-formed somatic embryos were transferred to MS medium containing 3% sucrose and 0.8% agar for shoot production. The embryos were elongated in MS medium supplemented with 1 mg L−1 gibberellic acid, 0.5 mg L−1 indole-3-butyric acid, 100 mg L−1 myo-inositol, and 3.76 mg L−1 riboflavin. Root regeneration was achieved on MS medium containing 7.9 mg L−1 phloroglucinol and supported with vermiculite after 4 days of cultivation on ½ MS medium with 2 mg L−1 indole-3-butyric acid. After the rooting phase, in vitro plantlets were acclimatized in peat moss soil.

Symplasmic isolation marks cell fate changes during somatic embryogenesis.

Cell-to-cell signalling is a major mechanism controlling plant morphogenesis. Transport of signalling molecules through plasmodesmata is one way in which plants promote or restrict intercellular signalling over short distances. Plasmodesmata are membrane-lined pores between cells that regulate the intercellular flow of signalling molecules through changes in their size, creating symplasmic fields of connected cells. Here we examine the role of plasmodesmata and symplasmic communication in the establishment of plant cell totipotency, using somatic embryo induction from Arabidopsis explants as a model system. Cell-to-cell communication was evaluated using fluorescent tracers, supplemented with histological and ultrastructural analysis, and correlated with expression of a WOX2 embryo reporter. We showed that embryogenic cells are isolated symplasmically from non-embryogenic cells regardless of the explant type (immature zygotic embryos or seedlings) and inducer system (2,4-dichlorophenoxyacetic acid or the BABY BOOM (BBM) transcription factor), but that the symplasmic domains in different explants differ with respect to the maximum size of molecule capable of moving through the plasmodesmata. Callose deposition in plasmodesmata preceded WOX2 expression in future sites of somatic embryo development, but later was greatly reduced in WOX2-expressing domains. Callose deposition was also associated with a decrease DR5 auxin response in embryogenic tissue. Treatment of explants with the callose biosynthesis inhibitor 2-deoxy-D-glucose supressed somatic embryo formation in all three systems studied, and also blocked the observed decrease in DR5 expression. Together these data suggest that callose deposition at plasmodesmata is required for symplasmic isolation and establishment of cell totipotency in Arabidopsis.

Histocytological Study of Somatic Embryogenesis in the Tree Cinnamomum camphora L. (Lauraceae)

Histocytological studies were conducted on primary, secondary, and malformed embryos produced during somatic embryogenesis of Cinnamomum camphora L. to better understand its development. Exploring its callus types and structures provided a theoretical basis for clarifying the mechanism of somatic embryogenesis, which may shed light on the mechanism of zygotic embryogenesis. We used immature zygotic embryos as explants to induce somatic embryos, forming many embryogenic calli that differentiated into mature somatic embryos. Our results showed that somatic embryogenesis of C. camphora was similar to that of zygotic embryos. We have been dedifferentiated four types of callus. Compared with non-embryogenic cells, embryogenic cells had a closer arrangement, larger nucleus, thicker cytoplasm, more starch granules and easier to stain into black. Somatic embryogenesis had two pathways: direct (predominate) and indirect (rare). Embryogenic cells of C. camphora could have either an internal or external origin, the latter being primary, for which occurrence sites include epidermis and near-epidermis (little internally). Mostly arising from single cells, C. camphora follows two developmental pathways: single-cell equal as opposed to unequal, wherein both divide to form multi-cell proembryos. However, multicellular origins can occasionally occur and feature physiological isolation during somatic embryo development. This development has four embryo stages: globular, heart-shaped, torpedo, and cotyledon, with procambium cells apparent in globular embryos and late cotyledons forming “Y-shaped” vascular bundles. Secondary embryos were present in all stages, directly occurring on primary embryo’s germ and radicle end surfaces. We conclude that secondary and primary embryos of C. camphora undergo similar developmental processes. At the same time, conjoined cotyledon embryos and morphological abnormal embryos were found, with an internal origin more likely to generate abnormal embryos.&#x0D; &#x0D; *********&#x0D; In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 4, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue.&#x0D; *********

Plant regeneration by somatic embryogenesis in Pinus thunbergii resistant to the pine wood nematode

Pine wilt disease (PWD) is a severe threat to pine forests in East Asia. Screening and breeding of resistant varieties is a very effective way to prevent and control PWD; however, no reliable somatic embryogenesis system has yet been developed for the elite nematode-resistant Pinus thunbergii Parl. line. In this study, we studied the plant regeneration via somatic embryogenesis of nematode-resistant P. thunbergii. Initiation of embryogenic tissue was significantly affected by seed family (p = 0.017), immature zygotic embryo stage (p = 0.032), and initiation medium (p = 0.004). Seed family 37 was the most favorable female parent for initiation of P. thunbergii. Furthermore, the initiation rate increased from the pre-embryonic stage to the cleavage polyembryonic stage. The optimal medium was I2, containing 2,4-dichlorophenoxyacetic acid (9 μmol·L−1) and 6-benzyladenine (4.4 μmol·L−1). A statistically significant interaction between cell line and subculture time (24 months) was observed in the influence on proliferation rate, somatic embryo production, and percentage germination (p &lt; 0.001). In this study, the highest somatic embryo production was achieved using cell line 37-1 (1983 somatic embryos per gram fresh mass), with approximately 83.5% of somatic embryos germinating after transferring to germination medium, of which 77.6% converted into plantlets.

The application of different reproduction techniques for rare species waterlily tulip (Tulipa kaufmanniana Regel.) propagation under ex situ conditions

T. kaufmanniana is a threatened and endemic species of the Tien Shan Mountains with a complex of valuable ornamental features. However, the commercial usage of the plant is limited due to the restricted availability of the bulbs in nature, difficulties in overcoming seed dormancy and low efficiency of the species reproduction. Results of the investigation of the reproductive biology of this species in the culture conditions allowed us to characterize T. kaufmanniana as viable with successful seedage. In the work reported here the effect of low temperature on the proper development of embryos is addressed, and an attempt is made to clarify the effect of cytokinins (TDZ, BAP) and auxin (NAA) on the regeneration capacity of isolated T. kaufmanniana embryos for the adventitious shoots and bulblets formation. Direct shoot organogenesis was induced by the combined action of chilling and BAP or TDZ treatment. Among the variants tested, TDZ promoted the formation of adventitious bulbs in 48 % of the chilled embryos, compared to non-chilled embryos (0.0%) and embryos exposed to the same concentration of BAP (39%). The best culture condition for T. kaufmanniana embryos growth and bulblets formation consisted of chilling for 10 wk in the dark at 4 °C, 4 wk at 20 °C under 16 h light photoperiod and 10 wk at 4 °C in the dark. Sufficiently effective protocol for clonal micropropagation was developed for the first time for T. kaufmanniana by the use of immature zygotic embryos as primary explants.

Recurrent somatic embryogenesis and development of somatic embryos in Akebia trifoliata (Thunb.) Koidz (Lardizabalaceae)

A simple and effective protocol was established for recurrent somatic embryogenesis and plant regeneration in Akebia trifoliata (Thunb.) Koidz. Somatic embryos directly formed from the root zone of the immature zygotic embryos cultured on MS medium devoid of plant growth regulator (PGR). The induction frequency of immature zygotic embryos was 52.5%, and the mean number of somatic embryos reached up to 19.5. Secondary somatic embryos arose on the primary embryos at a high frequency (95.8%), and this process maintained in a recurrent way on PGR-free MS medium. The highest mean number of somatic embryos also appeared in the primary embryos which can reach up to 44.7. While both of the embryogenic potential and mean number of embryos per explant displayed a gradual diminution with subculturing. The addition of 0.5 mg l−1 6-benzyladenine in the 1/2 MS regeneration medium can significantly improve the frequency of somatic embryos convert to plantlets. The microscopic analysis revealed that the development process of somatic embryos via the globular, heart, torpedo and cotyledonal stages in A. trifoliata, the histological analysis showed that somatic embryos directly initiated from root epidermal cells and there are no vascular connections between the somatic embryos and maternal tissue. Regenerated plantlets acclimated successfully to greenhouse conditions. Approximately 65% plantlets survived and displayed no morphological characteristics differences with seed-derived plants. The simple and effective protocols established in this study will promote large-scale clonal propagation and genetic improvement of A. trifoliata. Somatic embryogenesis has never been reported in Akebia trifoliata (Thunb.) Koidz. The study showed the origin and development process of somatic embryos in Akebia trifoliata (Thunb.) Koidz.

How to shorten a plant breeding program? A case study with ornamental peppers

Abstract Plant breeding of ornamental peppers (Capsicum spp.) can be supported by biotechnological tools. The objective of this study was to evaluate the efficiency of an in vitro culture of immature zygotic embryos (IZE) to reduce the breeding cycle of ornamental pepper (C. annuum) in comparison to the conventional system. Three ornamental pepper genotypes were used: UFPB 001, UFPB 004, and UFPB 099. Embryos at 30 days after selfing were inoculated in MS ½-strength culture medium, and at the same time, seeds were placed to germinate in a commercial substrate. Approximately 215 days are required from selfing until fruit ripening in the conventional system, whereas the IZE system requires an average of 153 days, a decrease of approximately 30% per selection cycle, corresponding to 496 days considering 8 selfing cycles. A decrease in time, labor, and inputs makes the IZE system a suitable tool for shortening the breeding program of ornamentals peppers.

Effect of 2, 4-D and Picloram on Somatic Embryogenesis in Carica papaya var. P-7-9

Immature zygotic embryos of Carica papaya L. var. P-7-9 were inoculated on half-strength MS with different concentrations of 2,4-D and picloram alone and their combinations. The highest induction (80%) of somatic embryogenesis was MS supplemented with 2,4-D (7.0 mg/l) followed by 2,4-D (4.0 mg/l) + picloram (1.0 mg/l) but no embryogenesis was observed in auxin-free medium. The addition of osmoticum such as PEG and ABA significantly increased the maturation of somatic embryos. BAP, TDZ and NAA were used for shoot induction. Combination of 2.0 mg/l BAP and 0.5 mg/l NAA was found significantly better for shoot bud proliferation and 70% multiplication. The percentage of germinating embryos was enhanced significantly when the MS fortified with GA3 (0.5 mg/l). However, the high frequency of embryo germination and plantlets formation were obtained with 2,4-D (4.0 mg/l) + picloram (1.0 mg/l).&#x0D; Plant Tissue Cult. &amp; Biotech. 29(1): 25-32, 2019 (June)

In Vitro Regeneration of Sugar Palm (Arenga pinnata Wurmb Merr.)

Optimization of a protocol for in vitro regeneration of sugar palm (Arenga pinnata Wurmb Merr.) by direct organogenesis using the immature zygotic embryo and basal stem explants of in vitro raised seedlings has been developed. The explants were cultured on MS medium supplemented with different concentrations of PGRs, and the organogenic capability of the explants was investigated. Both explants were responsive, but the results were greatly dependent on the genotype and the culture medium composition investigated. After 8 weeks of culture, basal stem explant cultured on MS + 1.0 mg/L Kin + 2.0 mg/L NAA promoted optimum response for direct organogenesis (90%) with a total of 9 adventitious shoots and 25 roots. Subculturing of individual shoots in basal MS medium (MS0) optimized further development. Meanwhile, immature zygotic embryo explant cultured on MS + 2.0 mg/L BAP + 2.0 mg/L NAA promoted optimum root regeneration (70%), though MS medium containing 2.0 mg/L BAP + 1.0 mg/L GA3 and 1.0 mg/L AgNO3 promoted optimum shoots growth (total of 10 shoots) at the rate of 0.04%. In vitro rooting of the regenerated shoots was successfully obtained on MS + 3.0 mg/L IBA. Established in vitro plantlets with roots were hardened in soil: peat moss: perlite (2:2:1) mixture. Acclimatization of plantlets was successfully obtained with 70% seedlings survived after 4 month’s exposures under the light intensity of 50-100 µmol m-2s-1.

Development of embryogenic dogwood cultures and the regeneration of plants.

Somatic embryogenesis in flowering dogwood (Cornus florida) has been achieved, but not the regeneration of plants with active shoot growth. To improve plant regeneration, eight media treatments were tested for induction of somatic embryogenesis from immature zygotic embryos. Somatic embryogenesis was obtained on three media containing the plant growth regulators (PGRs) 0.1 mg l-1 picloram, 2.0 mg l-1 2,4-dichlorophenoxyacetic acid, or 0.1 mg l-1 indole-3-butyric acid (IBA). Somatic embryogenesis was also induced on Woody Plant Medium without PGRs. Overall, 92% of the somatic embryos examined converted into plants with active root and shoot growth. This is the first report of somatic embryo-derived plants of C. florida that have active shoot growth and that could be transferred to soil. Embryogenic suspensions were established from IBA-treated cultures that could serve as a target for C. florida bioengineering.