Culture Of Zygotic Embryos Research Articles

In vitro germination of zygotic embryos excised from cryopreserved endocarps of queen palm (Syagrus romanzoffiana (Cham.) Glassman)

Queen palm (Syagrus romanzoffiana [Cham.] Glassman) is a palm species best known as an ornamental tree for urban landscaping, but recently, it has been evaluated as a potential crop for biofuel production. The objective of the present work was to establish a cryopreservation technique for queen palm to ensure long-term conservation of this species. The cryopreservation protocol consisted of direct immersion in liquid nitrogen (LN) of whole endocarps with water contents ranging from 5.5 to 10.9%, followed by slow thawing at room temperature (25 ± 2°C) excision and in vitro culture of zygotic embryos. Viability of zygotic embryos isolated from endocarps with different water contents was evaluated before (control) and after freezing in LN using in vitro culture on Woody Plant Medium (WPM) medium. Germination percentages of zygotic embryos isolated from endocarps stored in LN varied from 84 to 93%, whereas those isolated from controls ranged from 55 to 71%. Germination rates were significantly higher for zygotic embryos excised from cryopreserved endocarps. The water content of control or frozen endocarps did not have a significant effect on germination percentages of zygotic embryos. Zygotic embryos excised from endocarps following cryopreservation in liquid nitrogen developed into normal plantlets after in vitro culture. The technique tested is simple, efficient, and can be used in plant gene banks as a routine approach for long-term conservation of queen palm germplasm.

Estrategias para mejorar la germinación de semillas de Calyptrogyne ghiesbreghtiana (Linden y H. Wendland)

Because of the phytogenetic resources extraction and conservation interest, germination induction tests were carried out in the Calyptrogyneg ghiesbreghtiana (Linden & H. Wendland) species, known as guanito de taliz, a threatened species of the Arecaceae family. Greenhouse germination tests were performed using four treatments: immersion in 0.3% H 2 O 2 , 0.02% KNO 3 , 1% GA3 and distilled water soaking, all of them applied for a 24 h period on seeds without epycarpium; a control treatment on seeds with epycarpium was included. Another alternative used was the in vitro germination induction, using whole seeds and isolated embryos; to do so, each flask with three embryo or seeds in MS medium with sucrose added to 3% without growth regulators and solidified with Phytagel (2 gL-1). This was considered as an experimental unit. By evaluating the effect of chemical substances in C. ghiesbreghtiana seed germination, It was found that immersion in 0.3% H 2 O 2 solution resulted in the highest greenhouse germination percentage (84.59%), statistically different from the rest of the methods used and the control treatment. With regard to the rescue and culture of zygotic embryos under in vitro conditions, the best answer was observed with the complete isolated of embryos. In both strategies, it was really important to carry out the experiments with fresh seeds with no more than two weeks from collection, due to the loss of cell viability. The use of H 2 O 2 , under greenhouse conditions, implies an economical option for nurserymen and it can contribute to the production of plants for reforestation, as well as the use of embryo rescue technique to support conservation and wise use of the species.

Влияние аскорбиновой кислоты и глутатиона на индукцию соматического эмбриогенеза Picea pungens Engelmann

Effect of exogenic antioxidants – ascorbic acid (AA) and reduced glutathione (GSH) – on initiation and proliferation processes of somatic embryos of blue spruce (Picea рungens) in vitro embryos culture in dependence of stage development, genotype of donor trees, medium composition was shown using a cytological analysis. Initiation of somatic embryogenesis was carried out on basic media ½ DSR and ½ LV supplemented with AA (0 and 300 mg/l) and/or GSH (0 and 300 mg/l). The highest frequency of callus formation (from 60 up to 100 %) was obtained when establishing in zygotic embryo culture at the cotyledon stage. Formation of embryonal-suspensor masses (ESM) was detected at the initiation stage with the help of cytological analysis. The highest frequency of ESM formation achieved 8,4 % on ½ DSR medium and 28 % on ½ LV medium. Active proliferation was observed in only one of the genotypes studied. Application of antioxidants stimulated the frequency both of callus formation and ESM, however, AA and GSH affected on development of somatic embryos in different ways. Application of AA at the initiation and proliferation stages promoted formation of somatic embryos; GSH, on the contrary, reduced their development. Formation of the great number of somatic embryos was achieved with optimal combination of antioxidants: culture incubation at the initiation stage with AA and addition of GSH in culture medium at proliferation stage.

Phenolic Compositions of Litchi Shoot Tips and Zygotic Embryos Collected in Different Months and Their Effects on the Explant Browning and Its Control

In the research it was examined the changes in total phenolic contents and seven major phenolic compounds (gallic acids, +(–) catechin, catechol, Chlorogenic acid, o-coumaric acid, rutin and quercetin) of two litchi cultivars (Purbi and Bedana) shoot tips and fruits (for zygotic embryos) collected in different months, in order to determine their effects on the explants browning during establishment stage of shoot tip culture. The concentrations of phenolic compounds varied depending on the cultivars and the months. Phenolic compounds showed various correlation coefficients with the explants browning. Total phenolic content and some individual phenolic compounds including +(–) catechin, catechol, gallic acid, chlorogenic acid and rutin quantified in this study showed significant positive correlations with the explants browning, while o-coumaric acid and quercetin did not exhibit any significant one. According to our results, explants browning are affected by the phenolic compounds at different ranges. In both litchi cultivars, shoot tips and fruits (for zygotic embryos) collected in March exhibited the lowest explants browning during the establishment stage as compared to those collected in the other months. So it may be possible to increase the success of shoot tip and zygotic embryo culture with the selection of the most suitable terms of explants collection. Browning of explants could be controlled by the use of antioxidants both in semi-solid and liquid culture.

Ecophysiological, anatomical and biochemical aspects of in vitro culture of zygotic Syagrus coronata embryos and of young plants under drought stress

Key message The in vitro culture of zygotic embryos is an efficient alternative to overcome dormancy ofSyagrus coronataand young plants showed high resilience during drought and rehydration.

High expression of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE coincides with initiation of various developmental pathways in in vitro culture of Trifolium nigrescens.

The aim of this study was to identify and examine the expression pattern of the ortholog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE gene from Trifolium nigrescens (TnSERK) in embryogenic and non-regenerative cultures of immature cotyledonary-stage zygotic embryos (CsZEs). In the presence of 1-naphthaleneacetic acid and N6-[2-isopentenyl]-adenine, the CsZE regenerated embryoids directly and in a lengthy culture produced callus which was embryogenic or remained non-regenerative. As revealed by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), the TnSERK was expressed in both embryogenic and non-regenerative cultures, but the expression level was significantly higher in embryogenic ones. An in situ RNA hybridization assay revealed that the expression of TnSERK preceded the induction of cell division in explants, and then, it was maintained exclusively in actively dividing cells from which embryoids, embryo-like structures (ELSs), callus or tracheary elements were produced. However, the cells involved in different morphogenic events differed in intensity of hybridization signal which was the highest in embryogenic cells. The TnSERK was up-regulated during the development of embryoids, but in cotyledonary embryos, it was preferentially expressed in the regions of the apical meristems. The occurrence of morphological and anatomical abnormalities in embryoid development was preceded by a decline in TnSERK expression, and this coincided with the parenchymatization of the ground tissue in developing ELSs. TnSERK was also down-regulated during the maturation of parenchyma and xylem elements in CsZE and callus. Altogether, these data suggest the involvement of TnSERK in the induction of various developmental programs related to differentiation/transdifferentiation and totipotent state of cell(s).Electronic supplementary materialThe online version of this article (doi:10.1007/s00709-015-0814-5) contains supplementary material, which is available to authorized users.

Alternative induction of de novo shoot organogenesis or somatic embryogenesis from in vitro cultures of mature zygotic embryos of passion fruit (Passiflora edulis Sims) is modulated by the ratio between auxin and cytokinin in the medium

Several reports on tissue culture-based techniques have been published for passion fruit (Passiflora edulis), an important tropical fruit crop. However, a system in which de novo shoot organogenesis (DNSO) or somatic embryogenesis (SE) can be induced from the same type of explant was not available so far. The present study describes an in vitro system that enables alternative induction of both morphogenetic pathways from P. edulis mature zygotic embryo. DNSO was observed when explants were cultured on Murashige and Skoog medium supplemented with high 6-benzyladenine (BA) to 2,4-dichloro-phenoxyacetic acid (2,4-D) ratios, such as 72.4 μM BA and 4.5 μM 2,4-D. Embryogenic calli were observed when low BA/2,4-D ratios, such as 4.5 μM BA and 72.4 μM 2,4-D were used. Morpho-anatomical characterization revealed that epidermal and sub-epidermal cells were involved with the regeneration process via both DNSO and SE pathways. These cells became meristematic and divided extensively to form protuberances after 12–15 days of culture in both systems. These protuberances led to the formation of either meristemoids or pro-embryogenic cell clusters, which differentiated into shoot buds or somatic embryos, respectively. The use of this system coupled with techniques that allow gene expression studies might greatly contribute to further studies on in vitro morphogenesis in Passiflora.

Morpho-histology and genotype dependence of in vitro morphogenesis in mature embryo cultures of wheat

Cellular totipotency is one of the basic principles of plant biotechnology. Currently, the success of the procedure used to produce transgenic plants is directly proportional to the successful insertion of foreign DNA into the genome of suitable target tissue/cells that are able to regenerate plants. The mature embryo (ME) is increasingly recognized as a valuable explant for developing regenerable cell lines in wheat biotechnology. We have previously developed a regeneration procedure based on fragmented ME in vitro culture. Before we can use this regeneration system as a model for molecular studies of the morphogenic pathway induced in vitro and investigate the functional links between regenerative capacity and transformation receptiveness, some questions need to be answered. Plant regeneration from cultured tissues is genetically controlled. Factors such as age/degree of differentiation and physiological conditions affect the response of explants to culture conditions. Plant regeneration in culture can be achieved through embryogenesis or organogenesis. In this paper, the suitability of ME tissues for tissue culture and the chronological series of morphological data observed at the macroscopic level are documented. Genetic variability at each step of the regeneration process was evaluated through a varietal comparison of several elite wheat cultivars. A detailed histological analysis of the chronological sequence of morphological events during ontogeny was conducted. Compared with cultures of immature zygotic embryos, we found that the embryogenic pathway occurs slightly earlier and is of a different origin in our model. Cytological, physiological, and some biochemical aspects of somatic embryo formation in wheat ME culture are discussed.

Embryo initiation from Pinus sibirica megagametophytes in in vitro culture

Megagametophytes of Siberian pine were cultured on an in vitro culture medium 1/2 LV supplemented with growth regulators 2,4-dichlorophenoxyacetic acid (2,4-D) and benzylaminopurine (6-BAP) to form embryos. The competency of somatic cell of explants to embryogenesis manifested itself in an organized growth and polarity. A coenocyte consisting of long vacuolated cells was formed in the megagametophyte culture. Then, the migration of the nuclei to one of the poles of the cell, their division, and formation of embryoids was observed. The megagametophyte culture of the Siberian pine differed from the zygotic embryo culture by the absence of asymmetric division in the vacuolated cell.

Manifestation of embryogenic potential in culture of zygotic embryos of Quercus robur L.

For the initiation of somatic embryogenesis early cotyledonary stage of zygotic embryo explants (from 15th July until late August) was suitable. The highest frequency of differentiation of somatic embryos was obtained on cotyledons of zygotic embryos cultured on basal modified medium MS (with 1/2 concentration macronutrients) or WPM medium containing 500 mg•l<sup>-1</sup> glutamine, proline and casein hydrolysate and supplemented with 2,4-D (1,0-2,0 mg•l<sup>-1</sup>) and BAP (0,5-1,0 mg•l<sup>-1</sup>). The development of somatic embryos was direct and indirect and the process was continuous over a long period. Primary somatic embryos were able to produce secondary embryos. Repetitive somatic embryogenesis led to the proliferation of a large number of new somatic embryos on their cotyledons, hypocotyl or radicula. The process of embryo differentation is asynchronous - various stages of somatic embryos could be observed in embryogenic culture. A somatic embryo conversion was rare on tested media. Embryo germination occured on medium containing BAP (0,1 mg•l<sup>-1</sup>) or on medium with ABA and GA<sub>3</sub> (each 0,2 mg•l<sup>-1</sup>) after a previous culture on WPM medium without plant growth regulators supplemented with sorbitol (6%). The embryo germination occurred also on WPM medium with 0.2 mg•l<sup>-1</sup> BAP when cultures were mantained at 2<sup>o</sup>C for 4 weeks. Only 8 somatic embryos developed into plantlets. Their transplantation to <em>in vivo</em> conditions was unsuccessful.

Optimization of culture media for in vitro Zygotic embryo culture of Senna alata L. Roxb –An Ethnomedicinal plant

Senna alata L. Roxb, is an ethnomedicinal plant belonging to Fabaceae family. The plant is known to possess potent medicinal properties include laxative, antitumor, antimutagenic, antioxidant and antifungal. Seeds of S. alata possess a hard seed coat which acts as a barrier for seed germination. Zygotic embryos were cultured on various media (half and full - strength Murashige and Skoog's medium, Gamborg's medium) with different concentrations of sucrose(10-30g/L) and plant growth regulators (PGRs). Maximum percentage of embryo germination and healthy plantlet formation were observed on MSO (Murashige and Skoog basal) medium containing 20 g/L sucrose without PGRs. Multiple shoot formation was observed on MS (Murashige and Skoog's) medium containing 20g/L sucrose supplemented with 2 mg/L BAP. The formed seedlings were acclimatized in sterile vermiculite: garden soil (1:1) and later shifted to field conditions. The newly formed plantlets resembled the donor plant phenotypically. This protocol can be used for multiplication and conservation of the species.

Sustainable production of azadirachtin from differentiated in vitro cell lines of neem (Azadirachta indica)

Azadirachtin has high industrial demand due to its immediate application as an ecofriendly, biodegrad- able biopesticide and also due to its various other significant bioactivities. To date, the only commercially feasible way to produce azadirachtin is extraction from seeds, but their availability is very limited as the tree flowers only once a year and only one-third of the fruits are collected due to operational problems. Further, due to the strict out- breeding nature of the plant, the seeds are highly heterozygous, resulting in inconsistent metabolite production. There- fore, in the present study, to achieve sustainable production of azadirachtin, dedifferentiated and redifferentiated calli derived from various explants of neem—zygotic embryo, leaf and ovary—were investigated for their potential to bio- synthesize azadirachtin. High-performance liquid chromatography analysis of theinvitro cell lines showed the presence of azadirachtin in all the samples tested, the content of which in cultured cells varied with explant source and cell dif- ferentiation response. The presence of azadirachtin in samples was further confirmed by positive electrospray ionization mass spectroscopy. The zygotic embryo cultures of neem accumulated much higher amounts of azadirachtin than leaf and ovary cultures. Furthermore, organizedinvitro callus cultures (redifferentiated) supported higher azadirachtin bio- synthesis, while unorganized callus cultures (dedifferentiated) supported the least. The maximum azadirachtin content of 2.33 mg g 21 dry weight was obtained from redifferentiated immature zygotic embryo cultures.

Somatic embryogenesis in Alnus glutinosa (L.) Gaertn

Induction of somatic embryos and plant regeneration was demonstrated for the first time in Alnus glutinosa. Somatic embryos were initiated from zygotic embryos collected 1–3 weeks post-anthesis (WPA), i.e., when they were at globular or early cotyledonary stage and were 0.5–1 mm in length. Induction frequency (16.6 %) and the mean number of somatic embryos (4.5 embryos/explant) were highest after culture of zygotic embryos, collected at 3 WPA, on Murashige and Skoog medium (MS) supplemented with 0.9-μM 2,4-dichlorophenoxyacetic acid and 2.22-μM benzyladenine (BA). No embryogenic induction was observed on medium with BA alone. Initial somatic embryos differentiated indirectly from callus tissue formed at the surface of the zygotic embryos. Embryogenic competence was maintained by secondary embryogenesis, which was affected by explant type, plant growth regulators and genotype. Secondary embryogenesis was induced by culture of small groups of whole somatic embryos or isolated cotyledon explants on medium consisting of MS medium (half-strength macronutrients) supplemented with 0.44-μM BA. Histological study of isolated cotyledon explants revealed that secondary embryos developed directly from differentiated embryogenic tissue on the surface of cotyledons. Somatic embryos at successive stages of development, including cotyledonary-stage embryos with shoot and root meristems, were evident. For plantlet conversion, somatic embryos were transferred to maturation medium supplemented with 3 % maltose, followed by 6 weeks of culture in Woody Plant Medium supplemented with 0.44-μM BA and 0.46-μM Zeatin (Z). This novel protocol appears promising for mass propagation, conservation and genetic transformation of black alder.

High-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis

This study reports high-frequency plant regeneration from immature zygotic embryo cultures of Houttuynia cordata Thunb via somatic embryogenesis. Numerous green globular structures were directly formed on the surfaces of cotyledons and radicles from 2-week-old immature zygotic embryos at a frequency of 42.1 % when cultured on Murashige and Skoog (MS) medium supplemented with 2 mg l−1 of α-naphthaleneacetic acid (NAA) and 1 mg l−1 of 6-benzyladenine (BA). In comparison, white globular structures and pale-yellow calluses were formed simultaneously at a frequency of 28.3 % when cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). The pale-yellow calluses were transferred to MS liquid medium supplemented with 2,4-D to establish embryogenic cell suspension cultures consisting of round, isodiametric cells that formed cell aggregates. Upon plating of these cell aggregates on half-strength MS medium without growth regulators under light conditions, cell aggregates gave rise to numerous globular embryos at a frequency of 56 %. Of the globular embryos, 15 % were successfully converted into cotyledonary embryos when cultured on half-strength MS medium under light conditions. The plant regeneration system of H. cordata established in this study will be useful for the selection, genetic transformation, and mass proliferation of elite clones with medicinal potential.

Morphological and agronomical characteristics of coconut (Cocos nucifera L.) palms produced from in vitro cultured zygotic embryos

In this study, we compared the evolution of morphological and agronomical characteristics of coconut (Cocos nucifera L.) palms produced from in vitro cultured embryos and from seeds over an 8-yr period. At the end of the second year after planting, the height and root collar diameter of plants originating from in vitro cultured embryos were significantly lower than those originating from seeds. However, palms from both categories had the same number of leaves. When palms originating from in vitro plantlets and from seeds were observed later in their development, they were similar for most morphological characteristics measured, except for minor differences in inflorescence morphology, which were still present 8 yr after planting. The flowering pattern, bunch, and fruit production were similar between the two categories of palms. These results indicate that in vitro culture of zygotic embryos does not adversely affect further development of palms in natural conditions.

Embryo culture is an efficient way to conserve a medicinally important endangered forest tree species Strychnos potatorum

The present study reports a protocol for germination of Strychnos potatorum (ver. Tel. Chilla) using zygotic embryo culture as an embryo rescue method. A 100% germination rate was obtained by culturing the embryos on full-strength Murashige and Skoog’s medium (MS) containing 20 g/L sucrose in comparison to McCown and Lloyd’s Woody Plant Medium (WPM). Germination rates decreased when the sucrose concentration was lower or higher than 20 g·L−1. WPM/MS medium containing glucose at levels 30, 20, 15 g·L−1 showed a smaller percentage of germination and at quarter strength, WPM/MS medium with glucose did not respond. Multiple shoot formation was found at 1.0–2.0 mg/L BAP; 3.0 mg/L Kn; 2.0 mg/L TDZ on MS medium with 20 g·L−1 sucrose. Germination rates improved when the embryos were placed upright (vertically) in the medium. The in vitro germinated seedlings were acclimatized in a walk-in-chamber and maintained in the green house with the survival rate of 65%–75%. These plants were transferred to the field and were found to be phenotypically normal, healthy and similar to donor plants. This protocol will be useful to overcome seed dormancy and for rapid multiplication and conservation of S. potatorum using zygotic embryo culture.

Micropropagation of Pinus peuce

In Pinus peuce zygotic embryo culture grown on Gresshoff and Doy (1972; GD) basal medium, 2.22 μM benzyladenine (BA) was superior in promoting adventitious bud induction during 4 weeks comparing to kinetin or BA + kinetin. Shoot elongation was achieved on half-strength GD medium devoid of plant growth regulators and containing activated charcoal. Pulse treatment with 1 mM indole-3-butyric acid (IBA) for 2 h, followed by transfer to half-strength GD medium, produced the most efficient rooting. Rooted shoots were transplanted to the greenhouse and plantlets continued to grow and developed into phenotypically normal plants. Up to 10 plants per explant can be obtained within 36 weeks from culture initiation.

In vitro zygotic embryo culture of Pinus peuce Gris.: Optimization of culture conditions affecting germination and early seedling growth

This study reports a protocol for the germination and early seedling growth of Pinus peuce Gris. using zygotic embryo culture. In order to overcome seed dormancy and optimize organogenesis, the effect of nutritional, plant growth regulatory and physical factors on in vitro germination and growth of isolated mature zygotic embryos of P. peuce were investigated.

Crescimento e aspectos sintomatológicos na aclimatização de Ipê-roxo

Este trabalho teve por objetivo avaliar a influência do tipo de vedação e substrato na pré-aclimatização e os aspectos sintomatológicos na aclimatização de Ipê-roxo. Para pré-aclimatização, plantas de Ipê-roxo obtidas in vitro por meio de cultura de embriões zigóticos foram inoculadas assepticamente em tubos de ensaio contendo diferentes tipos de vedação (algodão, tampa plástica + parafilme e tampa plástica) e diferentes substratos (ágar, vermiculita e Plantmax®) suplementados com meio de cultivo WPM acrescidos de 1 gL-1 de carvão ativado, 10 mgL-1 de ácido cítrico e 30 gL-1 de sacarose. Para a aclimatização, plantas de Ipê-roxo obtidas in vitro foram transplantadas para tubetes de 56 cm³; contendo vermiculita e Plantmax®na proporção 2:1, previamente autoclavado a 121ºC e 1 atm, por 20 minutos. Após o transplantio, as plantas foram irrigadas com diferentes concentrações do meio MS (25%, 50%, 75%, 100% e 150%), sendo utilizada como controle água destilada. Após 30 dias, foram verificados efeitos do tipo de vedação e substrato na pré-aclimatização. O algodão pode ser recomendado quando se utiliza Plantmax®ou vermiculita como substrato. Na aclimatização, verificou-se que as plantas quando irrigadas com água destilada e concentrações < 50% apresentaram sintomas de deficiência nutricional. Foram observados sintomas semelhantes com a utilização de MS em concentração igual ou superior a 100%. A concentração 75% do meio MS foi a mais eficiente na manutenção do vigor nutricional de mudas de Ipê-roxo em processo de aclimatização.

In Vitro Culture of Immature Zygotic Mango Embryos and Plantlet Development

In vitro culture of immature embryos may assist mango breeding in the production of hybrid plant material. However, zygotic embryo culture techniques have not been successfully developed for mango. To recover in vitro zygotic plants through embryo culture, ‘Lippens’ and ‘Keitt’ were used as a source of model immature embryos. Excised embryos were incubated in a liquid maturation medium to test different culture systems and media composition. Subsequent germination allowed for the recovery of complete in vitro plantlets. Variables included during artificial embryo maturation, independently or through paired interactions, significantly affected all the parameters measured for embryo development and characterization of the plantlets. Main effects of culture system (i.e., static versus agitation) and coconut water supply (20%) were responsible for up to 85.5% of total treatment variation. Direct and inverse interactions observed between culture system and either coconut water supplement or sucrose content (45 or 60 g·L−1) contributed to define the best combination of factors to improve embryo growth and plant formation. Complete plantlets could be obtained at a frequency above 83% for both cultivars at the end of the in vitro phase at a developmental stage that allowed acclimatization to greenhouse conditions.