Immature Zygotic Embryos Research Articles

LEAFY COTYLEDON1, FUSCA3 expression and auxin treatment in relation to somatic embryogenesis induction in Arabidopsis

The expression pattern of the LEC1 and FUS3 genes during somatic embryogenesis in Arabidopsis explants (immature zygotic embryos) induced in vitro was analysed, using Real-time quantitative PCR (qRT-PCR). The analysis revealed differential expression of LEC1 but not FUS3 within a 30 day time course of somatic embryo development, and a significant auxin-dependent upregulation of LEC1 was found over the time course. In contrast to embryogenic culture, the level of LEC1 and FUS3 expression was noticeably lower in non-embryogenic callus of Col-0 and hormonal mutants (cbp20 and axr4-1) with low SE-efficiency. In addition, the expression profile of LEC1 and FUS3 was followed in the embryogenic culture derived from 35S::LEC2-GR explants. A significant increase of LEC1 but not FUS3 activity was observed under LEC2 overexpression induced in auxin-treated explants. The work provides further experimental evidence on LEC gene involvement in the embryogenic response in Arabidopsis somatic cells, and also implicates LEC1 function in more advanced stages of SE culture in relation to somatic embryo differentiation and development.

Influence of AgNO3 on somatic embryo induction and development in Manchurian ash (Fraxinus mandshurica Rupr.)

In this present study, we explored the effects of silver nitrate (AgNO 3) on somatic embryo induction and the development from immature zygotic embryos in Manchurian ash ( Fraxinus mandshurica Rupr.). AgNO 3 played a minor role on in vitro embryo induction frequency and in the number of somatic embryos per explant. However, 1 mg L –1 AgNO 3 enhanced synchronization and significantly inhibited abnormal somatic embryo formation suggesting that AgNO 3 might serve an important function in controlling the development of somatic embryos in Manchurian ash. Our results provided foundation for a future more efficient somatic embryogenesis and regeneration protocol.

In Vitro Somatic Embryogenesis in Some Oil Yielding Tropical Tree Species

Somatic embryogenesis was achieved in two oil yielding tropical tree species i.e. Simarouba glauca & Azadirachta indica using immature zygotic embryos as explants on Murashige and Skoog (MS) medium supplemented with 0.5 – 1.5 mg/l benzylaminopurine (BA) and 2.0 - 3.0 mg/l NAA (1-napthaleneacetic acid) or 2, 4-D (2,4-dichlorophenoxyacetic acid) and 3% sucrose. MS medium containing 1.0 mg/l BA and 2.0 mg/l NAA was noted to be the most effective in inducing friable embryogenic callus (FEC) in Simarouba glauca; the number of somatic embryos per culture varied in MS medium supplemented with 1.0 – 1.5 mg/l BA and 1.0 mg/l NAA. In Azadirachta indica, somatic embryos developed on MS medium supplemented with 0.5 mg/l BA and 1.5 – 2.0 mg/l 2,4-D which were in various shapes and sizes after the first subculture on MS medium supplemented with 0.25 mg/l abscisic acid. The somatic embryos which developed shoots were isolated and rooted in 1/2 strength MS medium supplemented with 0.25 mg/l abscisic acid and 2% sucrose. About 25% of embryos germinated within 20 days of culture in case of Simarouba glauca and 62% in Azadirachta indica. The somatic embryo-derived plantlets were transferred to the field after being hardened in the climate controlled hardening chamber.

Plant Regeneration in <i>Sorbus. pohuashanenesis</i> Hedl by Somatic Embryogenesis

Somatic embryogenesis was obtained by using immature zygotic embryos of S. pohuashanesis as explants and emblings were obtained. For induction of somatic embryos, immature zygotic embryos which 30 days old after pollination were cultured on solid MS medium with 1.0 mg•L-1 NAA, 0.1 mg•L-1 6-BA, 500 mg•L-1casein hydrolysate (CH) and 40 g•L-1 sucrose . Inducted somatic embryos were cultured in solid MS medium containing 500 mg•L-1CH and 40 g•L-1 sucrose. After 30 days of culture, many normal cotyledonary embryos were produced. Plantlets were regenerated when somatic embryos were transferred to MS medium with 30 g•L-1 sucrose. The somatic embryos germinated at a germination frequency of approximately 80%, but rate of the plantlets that successfully acclimated and continued growing was 40% in the greenhouse.

Somatic embryogenesis of selected spruce species (Picea abies, P. omorika, P. pungens 'Glauca' and P. breweriana)

Somatic embryogenesis was studied in four spruce species (<em>Picea abies</em>, <em>P. omorika</em>, <em>P. pungens</em> 'Glauca' and <em>P. brewenana</em>) to determine if this method can be used for in vitro propagation of coniferous trees. The highest frequency of initiation of embryogenic tissue was obtained when mature zygotic embryos were used as explants. It ranged then from 10.8% (<em>P. brewenana</em>) to 23.75% (<em>P. omorika</em> and <em>P. pungens</em> 'Glauca'). The frequency of embryogenic tissue initiation was strongly affected by medium composition, i.e. addition of appropriate auxins (2,4-D, NAA, Picloram) and sucrose concentration (10-20 g<sup>-1</sup>"1). A lower frequency was obtained in <em>Picea omorika</em> (10%) when megagametophytes (endosperms with immature zygotic embryos) were used as explants. No emryogenic tissue was produced from hypocotyls, cotyledons and needles. A satisfactory frequency was achieved with the use of somatic embryos of <em>Picea abies</em> (30%). The proliferation of embryogenic cell lines of spruces was affected by medium type. The experiments resulted in production of somatic plantlets of <em>P. abies</em> and <em>P. omorika</em>. This enables the application of this method of spruce micropropagation for genetic and breeding research or for nursery production.

Micropropagação de plântulas de erva-mate obtidas de embriões zigóticos

O objetivo deste trabalho foi avaliar o potencial de micropropagação de erva-mate (Ilex paraguariensis Saint Hilaire) a partir de plântulas assépticas oriundas de embriões zigóticos imaturos. Ápices caulinares foram inoculados em meio base, formado por ¼ da concentração dos sais do meio MS, com diferentes doses de 6-benzilaminopurina (BAP). Ápices caulinares foram enraizados em meio base com diferentes doses de ácido indolbutírico (AIB) e avaliado o efeito do AIB do meio de enraizamento in vitro na aclimatização das plantas. O meio base com 8,88µM de BAP foi o mais efetivo para a multiplicação, promovendo o maior número e comprimento de brotações adventícias. Ápices caulinares enraizaram em 30 dias de cultivo em meio base, acrescido de 7,38µM de AIB. A dose de AIB utilizada no enraizamento in vitro interfere na aclimatização das plantas. A técnica de micropropagação descrita pode ser utilizada para a produção massal de plantas de erva-mate.

Immature Zygotic Embryo Cultures of Arabidopsis. A Model System for Molecular Studies on Morphogenic Pathways Induced In Vitro

Immature Zygotic Embryo Cultures of Arabidopsis. A Model System for Molecular Studies on Morphogenic Pathways Induced In Vitro

국내 임목류 기내증식 연구현황 및 전망

Plant micropropagation techniques include bud cultures using apical or axillary buds, organogenesis through callus culture or adventitious bud induction, and somatic embryogenesis. In Korea Forest Research Institute (KFRI), the first tissue culture trial in woody plant was initiated from the bud culture of hybrid poplars (Populus alba x P. glandulosa) in 1978. Since then several mass propagation techniques have developed from conifer and hardwood species, resulting in allowing practical application to Poplars, Birches and some oak species. In addition, useful micropropagation and genetic resources conservation techniques were established in some rare and endangered tree species including Abeliophyllum distichum. Among various in vitro propagation techniques, somatic embryogenesis is known to be the most efficient plant regeneration system. Since the first somatic embryo induction was reported in Tilia amurensis by KFRI in 1986, various protocols for direct or indirect somatic embryogenesis systems have developed in conifer and hardwood species including Larix leptolepis, Pinus rigida x P. taeda F1, Kalopanax septemlobus and Liliodendron tulipifera, etc. However, most of these technologies have been developed using juvenile tissues, i.e. immature zygotic embryos or mature embryos. Therefore it has been difficult to directly application to tree breeding program due to their unproven genetic background. Recently remarkable progresses and new approaches have been achieved in mature tree somatic embryogenesis. In this article we reviewed several micropropagation techniques, which have been mainly developed by KFRI and recent international progresses.

Lodgepole pine: the first evidence of seed-based somatic embryogenesis and the expression of embryogenesis marker genes in shoot bud cultures of adult trees

Of the various alternatives for cloning elite conifers, somatic embryogenesis (SE) appears to be the best option. In recent years, significant areas of lodgepole pine (Pinus contorta) forest have been devastated by the mountain pine beetle (MPB) in Western Canada. In an attempt to establish an SE propagation system for MPB-resistant lodgepole pine, several families displaying varying levels of resistance were selected for experimentation involving shoot bud and immature seed explants. In bud cultures, eight embryogenic lines were induced from 2 of 15 genotypes following various treatments. Genotype had an important influence on embryogenic culture initiation, and this effect was consistent over time. These lines were identified by microscopic observation and genetic markers. Despite the abundance of early somatic embryos, the cultures have yet to develop into mature embryos. In contrast, immature zygotic embryos (ZEs) cultured from megagametophytes initiated SE at an early dominance stage via nodule-type callus in 1 of 10 genotypes. As part of the study, putative embryogenesis-specific genes, WOX2 (WUSCHELL homeobox 2) and HAP3A, were analyzed in cultures of both shoot bud explants and ZEs. On the basis of these analyses, we postulate that PcHAP3A was expressed mainly in callus and may be involved in cell division, whereas WOX2 was expressed mainly in embryonal mass (EM)-like tissues. The findings from this study, based on molecular assessment, suggest that the cell lines derived from bud cultures were truly EM. Moreover, these experimental observations suggest that PcWOX2 could be used as an early genetic marker to discriminate embryogenic cultures from callus.

Induction of somatic embryogenesis and plant regeneration in the tropical timber tree Spanish red cedar [Cedrela odorata L. (Meliaceae)

Spanish red cedar (Cedrelaodorata L.) is a tropical timber tree native to the Americas from southern Mexico to northern Argentina. Commercial plantations are scarce and, consequently, natural populations are overexploited. Traditional propagation practices for the establishment of large-scale plantations have had limited success in this species due to the relative scarcity of seeds, its broad genetic diversity and the lack of domesticated varieties. In vitro clonal propagation provides an effective method to overcome this situation and increase the yield of commercial plantations through the rapid multiplication of elite materials. Somatic embryogenesis (SE) is one of the most promising strategies for tree propagation due to the possibility of producing artificial seeds, the ability to store and rapidly mobilize germplasm and the opportunity for genetic manipulation. We report here the induction of indirect SE in C. odorata from calli derived from immature zygotic embryos after 12 weeks of culture. Macroscopic, histological, and scanning electron microscopic analyses of the calli revealed the presence of embryogenic cell clusters that formed cotyledonary embryos with clear bipolar structures and no vascular connections with the mother tissue. Different media preparations containing combinations of diverse auxins and cytokinins are known to have different effects on the type and frequency of embryogenic structures. Embryo conversion was achieved using an MS-based medium [Murashige T, Skoog F (1962) Physiol Plant 15:473–497, 1962] supplemented with abscisic acid, and transfer to soil was successful at a rate of 75%. The method described here provides a basis for optimizing the clonal propagation and genetic manipulation of this valuable species.

Somatic embryogenesis in four tree legumes.

Somatic embryogenesis was achieved in four leguminous tree species, that is, Acacia catechu, Acacia arabica, Hardwickia binata, and Dalbergia sissoo using immature zygotic embryos as explants on Murashige and Skoog (MS) medium supplemented with 0.25–1.0 mg/l Kn (kinetin) and 2.0–3.0 mg/l 2,4-D (2,4-dichlorophenoxyacetic acid) or NAA (1-napthaleneacetic acid) and 3% sucrose. MS medium containing 2.0 mg/l 2,4-D and 1.0–1.5 mg/l Kn was noted to be most effective in inducing friable embryogenic callus (FEC); the number of somatic embryos per culture varied in MS medium supplemented with 1.0–2.0 mg/l 2,4-D or NAA and 0.25–1.5 mg/l kinetin. The maximum number of somatic embryos was obtained in MS medium containing 1.5–2.0 mg/l 2,4-D or NAA and 1.0–1.5 mg/l kinetin; proliferation of embryogenic calli was enhanced in cultures having 1.0–2.0 mg/l 2,4-D, 1.0–1.5 mg/l kinetin, and 400–600 mg/l L-Proline. The somatic embryos in various shapes and sizes after the first subculture on MS medium supplemented with 0.1 mg/l IAA and 0.25 mg/l BA; developed shoots and rooted in 1/2 strength MS medium supplemented with 0.1 mg/l IBA or IAA. The somatic embryo-derived plantlets were transferred to the field after being hardened in the climate-controlled hardening chamber.

Plant regeneration using immature zygotic embryos of Tribulus terrestris

The genus Tribulus is the source of a number of steroidal saponins and other bioactive compounds which are of medicinal and pharmaceutical importance and plant regeneration of Tribulus terrestris has been reported. The objective of this study was to evaluate the potential of immature zygotic embryos of Tribulus terrestris as an explant for plant regeneration. Embryos were cultured on MS medium supplemented with 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and thidiazuron (TDZ), alone or in combination and callus and shoot or embryo formation evaluated. With 2.5 mg/l NAA or 2,4-D, callus formation frequency was 100% but 57% with 2.5 mg/l TDZ. The combination of 2.5 mg/l TDZ and NAA or 2,4-D also elicited callus formation frequency of 100%. The callus formation frequency was lower with lower levels of these growth regulators. On a medium with 0.5 mg/l TDZ, 17.4% of the 2,4-D-derived callus (2.5 mg/l), developed embryo-like structures and this increased to 37.3 and 41.4% respectively, when TDZ was combined with 0.5 mg/l indole-3-butyric acid (IBA) or 2,4-D. Both shoot formation and embryo-like structures developed in cultures with 2.5 mg/l TDZ, alone or in combination with 0.5 mg/l IBA or 2,4-D. The optimum sucrose level for morphogenetic response of embryo-derived callus was between 5.0 and 7.5%. Embryo-like structures were also observed when the 2,4-D-derived callus was cultured in a liquid containing benzyladenine (BA) and IBA. Plants were regenerated from both embryo-like structures and shoot buds on solid MS medium containing 0.2 mg/l IBA and rooted plantlets were transferred to soil.

Somatic embryogenesis of holm oak (Quercus ilex L.): ethylene production and polyamine content

The production of ethylene and the endogenous content of polyamines (PAs) have been recorded during the early development, maturation and germination of holm oak (Quercus ilex L.) somatic embryos. Ethylene production was high in embryogenic callus, immature somatic embryos and in explants showing secondary embryogenesis, while it was lower in mature and germinating somatic embryos. A higher ethylene production was also associated to the process of secondary embryogenesis. The exogenous application of 1-amino-1-cyclohexane carboxylic acid was not significantly effective on the production of ethylene by holm oak somatic embryos. Total PAs were more abundant in embryogenic callus and in both somatic and zygotic immature embryos, decreasing later on in the mature and germination phases. Immature somatic embryos of holm oak and immature zygotic embryos contain high levels of spermidine (Spd), which decreased during maturation and germination. Spermine (Spm) concentration was lower than that of Spd. Spm was more abundant in embryogenic callus and immature zygotic embryos than in mature embryos. Ethylene production did not seem to interfere with PA metabolism.

Morpho-anatomical characterization of embryogenic calluses from immature zygotic embryo of peach palm during somatic embryogenesis

The objective of this study was to morpho-anatomically characterize nodular embryogenic calluses from zygotic embryos of peach palm during the induction of somatic embryogenesis. Immature zygotic embryos were pre-treated in MS medium added to Picloram and 2,4-D (25 µM) and BAP (0, 5, 10 µM). After three months, primary calluses were transferred to MS induction medium added to Picloram and 2,4-D (450 µM). After six months, the embryogenic calluses were then histologically analyzed and cultivated in the maturation medium. The competent tissues of the zygotic embryos differentiated embryogenic calluses under action of both Picloram and 2,4-D auxins (450 µM), where the presence of multi-granular structures were observed. Histological observations showed that in the nodular embryogenic calluses, the outlying parenchymal cells exhibit cellular characteristics of high mitotic activity. Differentiation of tracheal elements exists in embryogenic calluses connecting the callus to the explant. The evaluated cytokinin/auxin interaction influences the development of embryogenic calluses and globular structures.

Transgenic Leucaena leucocephala expressing the Rhizobium gene pydA encoding a meta-cleavage dioxygenase shows reduced mimosine content

The use of the tree-legume Leucaena leucocephala (leucaena), which contains high levels of proteins in its foliage, is limited due to the presence of the toxic free amino acid mimosine. The goal of this research was to develop transgenic leucaena with reduced mimosine content. Two genes, pydA and pydB, encoding a meta-cleavage dioxygenase (EC 1.13.11.2) and a pyruvate hydrolase (EC 3.7.1.6), respectively, from the mimosine-degrading leucaena symbiont Rhizobium sp. strain TAL1145, were used to transform leucaena. These bacterial genes were sequence-optimized for expression in leucaena and cloned into the plant binary vector pCAMBIA3201 for Agrobacterium tumefaciens-mediated transformation. Using immature zygotic embryos as the start explant material, six pydA and three pydB transgenic lines were developed. The presence and expression of the bacterial genes in the transgenic lines were verified by PCR, reverse transcriptase PCR, and Southern analyses. HPLC analyses of the transgenic plants determined that the mimosine contents of the pydA-expressing lines were reduced up to 22.5% in comparison to the wild-type. No significant reduction in mimosine content was observed in the pydB-expressing lines. This is the first example of using a gene from a bacterial symbiont to reduce the toxicity of a tree-legume.

Development of embryo-like structures in the suspension cultures of flax coincides with secretion of chitinase-like proteins

Flax suspension cultures have been established from the callus induced from the cotyledons, hypocotyls, and immature zygotic embryos (iZE). The growth of flax suspension culture (expressed as a sedimented cell volume) was compared in both conditioned (by liquid from embryogenic Pinus nigra suspension culture) and non-conditioned media. Conditioning of media significantly increased the growth of the cell lines of hypocotyl and iZE origin; however, it had no promotive effect on embryogenic response of these flax liquid cultures. Formation of embryo-like structures (ELS), confirmed also histologically, has only been found in the cell line derived from iZE and cultivated in non-conditioned MS medium supplemented with 1 mg l−1 2,4-D. The process of ELS formation in this cell line was accompanied by the expression of the protein(s) with chitinolytic activity and molecular weight approximately of 25 kDa. The relationship between the formation of ELS and secretion of chitinase(s) is discussed.

10.1007/s11174-008-2004-2

Somatic embryogenesis was induced in Siberian larch by in vitro culturing zygotic embryos at different developmental stages. Cultures were grown in modified Murashige and Skoog medium supplemented with hormones 2,4-dichlorophenoxyacetic acid (2 mg/l) and 6-benzylaminopurine (0.5–1 mg/l). The success of somatic embryogenesis in this species depended on the tree genotype and developmental stage of embryos used for culturing. Somatic embryogenesis from immature zygotic embryos at the stage of cotyledon initiation was most active. After 5–10 days, such embryos formed the embryogenic tissue including two cell types—elongated highly vacuolated embryonic tubes and small embryonic cells. Somatic embryos were isolated from proliferating embryogenic tissues after 2 months of culture.

Regeneration of fertile plants from sunflower (Helianthus annuus L.): Immature embryo

SUMMARY Immature embryos from 15 sunflower genotypes (five restorers, five cytoplasmic male steriles, and five maintainers) were studied with the aim to shorten the seed to seed cycle, because seed maturation in sunflower takes 50- 60% of the life cycle duration (120-150 days). This technique allows the production of fertile plants from immature embryos by reducing the breeding cycle. Ten days after pollination immature embryos were dissected from seeds grown in field plants (SGFP) and were transferred to MS medium allowing shoot and root development for 5-10 days. Young plantlets were transferred to soil, developed to maturity, and were then self pollinated and set seed. The first cycle of immature embryo-raised plants (ERP) was obtained. When these plants were at the flowering stage, 10-day-old embryos were dissected and the 2nd cycle of ERP was obtained. The plants at the flowering stage for obtaining the 3rd cycle are in the growth chamber at the moment. The majority of cultured embryos developed into vigorous plantlets with 3-6 leaves. Out of the 1710 immature embryos, the average response of the explants was 93.1% (1591) showing morphogenesis with a minimum of 42.5 [N Record 109/Sanay 1-2(N)] and a maximum of 100% [(PR6404 ( cms ), Narmo Sanay 6-1 ( cms ), RIM 1-5 ( cms ), BGC0565 (N), N Record 109/Iscra (N), RHA 04, RHA 06, RHA 10, RHA 14, RHA 15]. Seventy percent of the developed plantlets had vigorous roots. They were transplanted into containers with a 1:1:2 peat: perlite: soil mixture (v/v) at 24 ± 2°C in 16 h/8 h (light/dark) in a growth chamber. Only 67.3% of them were grown to maturity, either self-pollinated or pollinated with maintainers and set seeds. The overall result was an average of 40-50 regenerated and matured plants per 100 immature zygotic embryos.

INFLUENCE OF POLYAMINE ON INDUCTION OF ADVENTIVE EMBRYONY IN PAPAYA (CARICA PAPAYA L.)

Somatic embryogenesis has been reported in different papaya cultivars across the world. However, all report suggests indirect embryogenesis in papaya with minimum of 3-6 months required for embryo formation. We have investigated role of polyamines (spermidine, spermine and putriscine) on direct embryogenesis on immature zygotic embryos excised from papaya cultivar Pusa Delicious. A brief exposure (2-4 weeks) of putriscine on induction medium (100 μM) augmented direct embryogenesis on explant. However, prolonged exposure of putriscine was found detrimental.

The somatic embryogenesis and plant regeneration from immature embryo of sweet corn inbred line

Synthetic seed consisting of somatic embryos enclosed in protective coating are a suitable tool for clonal mass propagation of elite plant varieties. The in vitro study was aimed to evaluate the optimizing medium for sweet corn somatic embryogenesis, synthetic seed production which leads to increasing germination and seed viability percentage. The in vitro study was aimed to evaluate the optimizing medium for sweet corn somatic embryogenesis, synthetic seed production which leads to increasing germination and seed viability percentage. Sweet corn (Zea mays var. saccharata) variety FAH01 embryogenic callus were derived from culturing immature zygotic embryos at 11 days after pollination on N6 medium that contained 2, 4-D 2 mg L-1 and sucrose 60 g L-1. Somatic embryos was developed after transferred embryogenic callus to N6 medium contained with 2 mg L-1 2, 4-D and 30 g L-1 sucrose. Sweet corn synthetic seed was produced by somatic embryos encapsulated into a protective calcium-alginate matrix with provides mechanical support, protection and is coated with a wax film to prevent desiccation. Synthetic seed were produced, it was found that when synthetic seed were treated with 60 g L-1 sucrose and stored at 15 ± 2 degree Celsius for 2 weeks, the percentage of germination of synthetic seeds were 42%, percentage of normal seedling was 91% and abnormal seedling was 8%, and they germinated for 8 - 9 days and could be produced normal planlet. When the synthetic seed were dehydrated by silica gel until remained 60% of their moisture content and then stored for 2 weeks, they could germinated at level 23%, which 83% of normal seedling and 17% of abnormal seedling. The survival ratio in sweet corn synthetic seed in this investigation indicated that there is still some more research required to increase number of the survival seeds and the optimum storage technique to prolong their viability is also needed Key words: Somatic embryo, synthetic seed, sweet corn, artificial seed, tissue culture.