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Question of unicellular origin of non-zygotic embryos in callus cultures
... An embryoid is identified by its polarity and claims single cell origin, it further should not be connected to the vascular supply of mother tissues at any stage of its life (Street and Withers 1974;Haccius 1978). Cabrera-Ponce et al. (2015) claimed somatic embryogenesis to be governed by cellular totipotency, quoting Haccius (1978) and Raghavan (1976) for their attributions to single cell or multi-celled origin, respectively. ...
... An embryoid is identified by its polarity and claims single cell origin, it further should not be connected to the vascular supply of mother tissues at any stage of its life (Street and Withers 1974;Haccius 1978). Cabrera-Ponce et al. (2015) claimed somatic embryogenesis to be governed by cellular totipotency, quoting Haccius (1978) and Raghavan (1976) for their attributions to single cell or multi-celled origin, respectively. ...
Santalum album L. is a commercially important tree that yields essential oil of high medicinal value. Regeneration research through organogenesis and embryogenesis has been documented but no report depicts comparative ontogeny of directly differentiating shoot buds (SB) and somatic embryos (SE). In the present study aseptic seedling derived hypocotyl segments (HC) and hypocotyl + root junction (HC + R) were used to induce SBs and SEs, respectively. Ontogenic differences between the structures were confirmed using scanning electron microscopy and histological analysis. MS medium containing 6-benzyladenine or BA (2.5 µM) produced highest number of direct SB, while MS + BA (7.5 µM) proved suitable for higher frequency of SE differentiation. The differentiating structures attained growth when transferred to MS medium containing a combination of BA and α-naphthalene acetic acid or silver nitrate (AgNO3). A combination of indole-3-butyric acid and silver nitrate (AgNO3) in half-strength woody plant medium and lesser osmotic concentration (2% sucrose), induced rhizogenesis.
... SE is a process by which a cell (Haccius 1978) or a group of cells (Williams and Maheswaran 1986) from somatic tissue form an embryo. In Agave, few reports exist on SE in economically important species (Table 17.1). ...
... The origin of somatic embryos can be unicellular (Haccius 1978) or multicellular (Williams and Maheswaran 1986). In agaves, a unicellular origin was observed in A. tequilana (Portillo et al. 2007), while a multicellular origin was observed in A. victoriae-reginae (Martínez-Palacios et al. 2003) and A. vera-cruz (Tejavathi et al. 2007). ...
This chapter describes the details of somatic embryogenesis of some species of the genus Agave. Various factors that influence the induction of the embryogenic process, such as explant type, growth regulator type, growth regulator concentration and genotype, are discussed. Furthermore, embryo development and conversion, as well as the first works using temporary immersion bioreactors, are also discussed.
... The early concept of somatic embryogenesis assumed that a somatic embryo originates from a single cell [97], which has been shown in various plants such as carrot [98]. However, the unicellular origin of somatic embryos is not the rule, and there are examples of the simultaneous occurrence of embryos with unicellular and multicellular origins in one regenerative system [99]. ...
Kohlrabi (Brassica oleracea var. gongylodes) is a vegetable cultivated for its edible stem tuber. Although valued for its nutritional properties and tolerance to abiotic stress, kohlrabi is one of the least studied brassicas. In this review, we summarize the results of our decade-long research on in vitro morphogenesis of kohlrabi, starting from 2013. Protocols for efficient in vitro regeneration with minimal requirements for external application of plant growth regulators (PGRs) have been developed, both by somatic embryogenesis (SE) and by de novo shoot organogenesis (DNSO). Efficient regeneration by direct SE was achieved using immature zygotic embryos as explants incubated on PGR-free media, and the production process was maintained in culture thanks to highly efficient secondary SE. Conversely, efficient regeneration by indirect DNSO was achieved using entire seedlings as explants and adding only cytokinin (CK) without the need for exogenous auxin. Comprehensive phytohormone analyses revealed that different exogenously applied CKs differentially affected the composition of endogenous phytohormones and induced changes in the expression of cell cycle-related genes and other genes involved in the organogenic response. The addition of high sucrose concentrations to the nutrient media failed to induce the formation of stem tubers in in vitro culture, but revealed a complex interaction with exogenously applied CKs, interfering with both the endogenous phytohormonome and the expression of organogenesis-related genes. Our work has provided substantial biotechnological advances in the field of in vitro regeneration of kohlrabi, as well as in understanding the underlying phytohormonal regulation. The review aims to introduce kohlrabi to the scientific community as a model system for both basic and applied research, while we continue seeking answers to the outstanding questions and trying to pave the way for the development of more resistant varieties.
... In borage somatic embryogenesis, globular structures appear to originate in two ways: from single cells (Fig. 2) or groups of cells (Fig. 4). These two patterns for the origin of somatic embryos have been described by Haccius (1978). Development from single cells closely mimics zygotic embryogenesis up to the globular stage (Fig. 2), as described in A tro p a belladon n a (Konar, 1972), D a u cu s ca ro ta L. (Street and Withers, 1974), T heobrom a ca c a o L. (Pence et al., 1980), and Sim m ondsia chin en sis (Link) C.K. Schneid. ...
Embryo sac formation in borage ( Borago officinalis L.) was of monosporic, Polygonum type. Zygotic embryogenesis resembled the Piperad type (Myosotis Variation) without a suspensor after the four-cell stage with free nuclear endosperm. Cotyledons were initiated 4 days after pollination (DAP) and grew rapidly until 12 to 14 DAP, with seed maturity at 18 DAP. There were two patterns of in vitro somatic embryo development from embryogenic callus: 1) from single cells, which proceeded through typical zygotic embryo stages; or 2) from groups of cells that developed as a meristem. The advanced stages of somatic embryogenesis were characterized by abnormal cotyledon and hypocotyl morphology and incomplete apical development.
... The somatic embryo may be produced either directly on the explant or indirectly from callus or cell suspension culture. For the first time, Haccius (1978) defined somatic embryogenesis as a nonsexual developmental process, which produces a bipolar embryo from somatic tissue. The first report of plantlet regeneration viain vitro somatic embryogenesis was in Daucus carota (Reinert, 1958;Steward et al., 1958). ...
... Validation of somatic embryogenesis is grounded on histological evidence that the structure is bipolar and has no vascular connection to the explant [97]. The most appropriate regeneration systems for transformation are direct or repetitive production of somatic embryos or de novo shoot organogenesis, which instigated from single cells of the epidermal layer [98,99]. ...
Study Objectives: An effort has been made to compute the optimal quantity of plant growth regulators to be added in culture medium and other physical factors exhibiting higher in vitro morphogenesis with 'elite' lines of sandalwood by culturing nodal segment. Results: Higher percentage of direct somatic embryogenesis, number(s) of somatic embryo per explant and plantlet regeneration via direct organogenesis were evidenced on MS medium augmented with a moderate concentration of TDZ (1.0 mgl-1) in combination with comparatively a lower concentration of NAA (0.5 mgl-1). A comparative higher concentration of BAP (1.0-2.0 mgl-1) in amalgamation with a lower concentration of NAA (0.5 mgl-1) encouraged frequency of indirect somatic embryogenesis. From culture media fortified with a greater concentration of BA at 4.0 mgl-1 in combination with a lower concentration of NAA, the proportion of organ development directly from the surface of cultured explants was recovered (0.5 mgl-1). Maximum plantlets regenerated via somatic embryogenesis (direct and/or indirect) on regeneration medium fortified with 2.0 mgl-1 TDZ in combination with 1.0 mg l-1 GA 3 , while plantlets in higher frequencies via indirect organogenesis was achieved with regeneration medium modified with relatively lower concentration of TDZ (1.0 mg l-1) in amalgamation with 0.5 mgl-1 GA 3 and 0.5 mg l-1 NAA. The plantlets were transferred to pots and hardened in Environmental Growth Cabinet and Net House during initial weaning period and shifted to field magnificently. Morphologically usual plants were obtained.
... Regeneration of putatively transformed cells and subsequent grafting of transgenic micro-shoots on rootstocks may shorten the juvenile period for flowering and fruiting [29]. The classical conception of somatic embryogenesis (SE) is based on the unicellular origin of somatic embryos [30], and this mode of somatic embryo development was the most frequently noticed in embryogenic cell suspensions of D. carota [31]. However, both a multicellular and a unicellular origin of somatic embryos in the same regeneration system is quite a common phenomenon, as was observed in several species, including Musa spp. ...
Agrobacterium-mediated transformation of epicotyl segment has been used in Citrus transgenic studies. The approach suffers, however, from limitations such as occasionally seed unavailability, the low transformation efficiency of juvenile tissues and the high frequency of chimeric plants. Therefore, a suspension cell culture system was established and used to generate transgenic plants in this study to overcome the shortcomings. The embryonic calli were successfully developed from undeveloped ovules of the three cultivars used in this study, “Sweet orange”-Egyptian cultivar (Citrus sinensis), “Shatangju” (Citrus reticulata) and “W. Murcott” (Citrus reticulata), on three different solid media. Effects of media, genotypes and ages of ovules on the induction of embryonic calli
were also investigated. The result showed that the ovules’ age interferes with the callus production more significantly than media and genotypes. The 8 to 10 week-old ovules were found to be the best materials. A cell suspension culture system was established in an H+H liquid medium. Transgenic plants were obtained from Agrobacterium-mediated transformation of cell suspension as long as eight weeks subculture intervals. A high transformation rate (~35%) was achieved by using our systems, confirming BASTA selection and later on by PCR confirmation. The results demonstrated that transformation of cell suspension should be more useful for the generation of non-chimeric transgenic Citrus plants. It was also shown that our cell suspension culture procedure was efficient in
maintaining the vigor and regeneration potential of the cells.
... These embryos or Germination of somatic embryos and adventitious shoots formation from embryogenic calluses. Embryos can be distinguished from adventitious shoots because they are bipolar, having both a shoot pole and a root pole, and do not have any vascular connections with the underlying parental tissues (Haccius 1978). Individual embryos were easily separated from the maternal tissues, as they were loosely attached at the radicle end to the mother explant (Fig. 1b,c,d). ...
Somatic embryogenesis and subsequent plant regeneration were established from hypocotyl and internode explants collected from in vitro-grown seedlings and in vitro-proliferated shoots, respectively. Somatic embryogenesis was significantly influenced by the types of auxin and cytokinin. Friable calluses with somatic embryos developed well in Murashige and Skoog basal (MS) medium supplemented with 0.8-8.8 μM 6-benzylaminopurine (BA) and 2.0-8.0 μM 2,4-dichlorophexoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The maximal frequency of embryogenic callus and somatic embryo formation were obtained when the MS medium was amended with 8.8 μM BA and 4.0 μM 2,4-D. The best embryo germination occurred in a hormone-free 1/2-MS medium. The highest percentage of shoot proliferation was observed in embryo-genic calluses in MS medium containing 2.0 μM BA and 1.0 μM NAA. In vitro-grown shoots were rooted in MS medium with 0.5-2.0 μM indole-3-butyric acid. Regenerants were transferred to vermiculite and successfully established under an ex vitro environment in garden soil.
... Embryos can be distinguished from adventitious shoots because they are bipolar, having both a shoot pole and a root pole, and do not have any vascular connections with the underlying parental tissues (18). Individual embryos were easily separated from the maternal tissues, as they were loosely attached at the radicle end to the mother explant (Fig. 1B). ...
A plant regeneration protocol was
established from hypocotyl explants of in vitro
grown seedlings of Asparagus officinalis and in
vitro-proliferated shoots, respectively through
either somatic embryogenesis or embryogenic
callus. Types of auxins and cytokinins play an
important role for producing somatic embryos.
Friable calli with somatic embryos developed well
in MS medium supplemented with 2.0-4.0 μM 6-
benzylaminopurine (BAP) and 1.0-4.0 μM 2,4-
dichlorophenoxyacetic acid (2,4-D), 1-
naphthaleneacetic acid (NAA) or indole-3-butyric
acid (IBA). The highest 96.3% embryogenic calli
and maximum number of 60.0 somatic embryos
formation were obtained from each explant when
the MS medium was fortified with 4.0 μM BAP
and 2.0 μM 2,4-D. The best embryo germination
occurred in 1.0 μM BAP supplemented ½ strength
MS medium. The highest 95.2% of shoot
proliferation was observed in embryogenic calli in
MS medium containing 2.0 μM BAP and 1.0 μM
IBA or NAA. In vitro-grown shoots were rooted in
½ strength MS medium with 0.5-4.0 μM IBA.
Regenerants were transferred to vermicompost and
successfully established under an ex vitro
environment in garden soil with 75.0% survival rate.
... These embryos or Germination of somatic embryos and adventitious shoots formation from embryogenic calluses. Embryos can be distinguished from adventitious shoots because they are bipolar, having both a shoot pole and a root pole, and do not have any vascular connections with the underlying parental tissues (Haccius 1978). Individual embryos were easily separated from the maternal tissues, as they were loosely attached at the radicle end to the mother explant (Fig. 1b,c,d). ...
Somatic embryogenesis and subsequent plant regeneration were established from hypocotyl and internode explants collected from
in vitro-grown seedlings and in vitro-proliferated shoots, respectively. Somatic embryogenesis was significantly influenced by the types of auxin and cytokinin.
Friable calluses with somatic embryos developed well in Murashige and Skoog basal (MS) medium supplemented with 0.8–8.8μM
6-benzylaminopurine (BA) and 2.0–8.0μM 2,4-dichlorophexoxyacetic acid (2,4-D) or α-naphthaleneacetic acid (NAA). The maximal
frequency of embryogenic callus and somatic embryo formation were obtained when the MS medium was amended with 8.8μM BA and
4.0μM 2,4-D. The best embryo germination occurred in a hormone-free 1/2-MS medium. The highest percentage of shoot proliferation
was observed in embryogenic calluses in MS medium containing 2.0μM BA and 1.0μM NAA. In vitro-grown shoots were rooted in MS medium with 0.5–2.0μM indole-3-butyric acid. Regenerants were transferred to vermiculite and
successfully established under an ex vitro environment in garden soil.
... La aparición del grupo de células embriogénicas indico el origen multicelular del embrión, como ocurre en Agave sisalana (Nikam et al., 2003), Agave veracruz (Tejavathi et al., 2007). Aunque otras referencias señalan el incuestionable origen unicelular de los embriones en varios cultivos (Street y Withers, 1974;Haccius, 1977), pero también ha quedado claro que el embrión puede tener un origen multicelular (Williams y Maheswaran, 1986). La masa proembriogénica de células formadas a partir de los grupos multicelulares tienen una orientación bipolar que carece de una conexión vascular con el tejido calloso (Figura 4E). ...
p> Background. Agave marmorata Roezl is a monocot plant of the Asparagaceae family, which is mainly used in the elaboration of the alcoholic drink called mezcal. This species is propagated by seeds or stolons (hijuelos), but its reproduction is slow because its maturation stage can last up to 35 years until flowreing. Therefore, efficient and alternative propagation methods should be used to ensure the permanence of the resource. Biotechnology, in particular plant tissue culture techniques, represents a viable alternative for the propagation of this species, however, until now, there are no previous reports on the spread of A. marmorata Roezl, via somatic embryogenesis. Objective. Evaluate various treatments to regenerate seedlings of this species through somatic embryogenesis. Methodology. Callus induction was obtained from mature seeds established in MS (Murashige and Skoog, 1962) at 25%, supplemented with vitamins L2 (Phillips and Collins, 1979), 5 mg L<sup>-1</sup> of 2,4-dichlorophenoxyacetic (2 , 4-D), 3 mg L<sup>-1</sup> of 6-benzyladenine (BA), 60 g L<sup>-1</sup> of sucrose, 8 g L<sup>-1</sup> of agar and adjusted to a pH of 5.7 ± 0.1. These calluses were sectioned into small portions weighing approximately 0.25 g and were incubated in an MS medium at 50% of their concentration with 30 g L<sup>-1</sup> of sucrose, 8 g L<sup>-1</sup> of agar and subjected to eight concentrations of two plant growth regulators (RCV): BA (0.0, 2.0, 6.0 and 10 mg L<sup>-1</sup>) and 2,4-D (0.0 and 5 mg L<sup>-1</sup>), alone or combined, giving a total of eight treatments. Those explants that responded to the embryogenic callus formation were subcultured to 50% MS medium, 30 g L<sup>-1</sup> of sucrose and gelled with 8 g L<sup>-1</sup> agar where two RCV were evaluated independently (0.1 mg L<sup>-1</sup> of 2,4-D or 3 mg L<sup>-1</sup> of AG<sub>3</sub>) and two culture conditions: 16 h light and 8 h dark and complete darkness, giving a total of 24 treatments. The variables evaluated in this work were: percentage of callus induction, percentage of embryogenic structures, number and growth of regenerated seedlings of A. marmorata Roezl at 255 ddic. Results. The best response was observed 120 days after starting the culture (ddic) in treatment five with 0.1 mg L<sup>-1</sup> of 2,4-D under light conditions where the pre-treatment was with 10 mg L<sup>-1</sup> of BA, obtaining 19.4 somatic embryos per explant. Maturation of somatic embryos in 50% MS medium was achieved with 30 g L<sup>-1</sup> of sucrose and 8 g L<sup>-1</sup> of agar, without RCV. The 100% of the regenerated seedlings survived and grew under greenhouse conditions. Implications. The results of this study contribute to a better understanding of the importance of a pretreatment with high concentrations of cytokinin (BA) and culture conditions, for the regeneration of Agave marmorata Roezl plants, via somatic embryogenesis. This can assist in the initiation of germplasm genetic improvement and in vitro conservation programs. Conclusions. The factors evaluated here were important in the induction and expression of embryogenic structures of this species, making possible the regeneration of seedlings of A. marmorata Roezl, from the formation of somatic embryos.</p
... At this point, the question of the origin of one or several cells for SE is directly related to the coordinated behavior of neighboring cells as a morphogenetic group [43,45]. According to several authors, a somatic embryo is defined as a new individual that arises from a single cell and has no vascular connection to the parent cells; multicellular origin seems to produce embryoids fused with parent cells over a wide area of the root pole of the axis region [13,46], while a unicellular origin is more likely to produce individual embryoids with a narrower structure similar to a suspensor [30,43,[45][46][47]. Therefore, the regeneration processes must be thoroughly studied in order to clarify and define whether a multicellular regenerate is a SE or an organ primordium. ...
Agave tequilana Weber cultivar 'Chato' represents an important genetic supply of wild severely in decline populations of 'Chato' for breeding and transformation programs. In this work, the indirect somatic embryogenesis and cryopreservation of Somatic Embryos (SEs) were investigated using the 'Chato' cultivar as a study case.
Methods:
Embryogenic calli were induced by the cultivation of 1 cm of young leaves from in vitro plants on MS semisolid medium supplemented with 24.84, 33.13, 41.41, 49.69, and 57.98 μM 4-amino-3,5,6-trichloro-2- pyridinecarboxylic acid (picloram) in combination with 2.21, 3.32, and 4.43 μM 6-benzylaminopurine (BAP). The origin and structure of formed SEs were verified by histological analysis. Cryopreservation studies of SEs were performed following the V-cryoplate technique and using for dehydration two vitrification solutions (PVS2 and PVS3).
Results:
The highest average (52.43 ± 5.74) of produced SEs and the Embryo Forming Capacity (estimated index 52.43) were obtained using 49.69 µM picloram and 3.32 µM BAP in the culture medium. The highest post-cryopreservation regrowth (83%) and plant conversion rate (around 70%) were achieved with PVS2 at 0 °C for 15 min.
Conclusion:
Our work provides new advances about somatic embryogenesis in Agave and reports the first results on cryopreservation of SEs of this species.
... During PLB development, histochemical assessment showed distinct morphological features. New PLBs developed from pre-existing ones resulting in a cyclic regeneration (Haccius 1978). Our results are in accordance with the previous findings (Liz 2013), in which C. tigrina leaves cultured in vitro displayed PLBs cyclic regeneration. ...
Cattleya tigrina is an endangered Brazilian native orchid with ornamental potential. Biotechnological tools based on tissue culture techniques are effective for its mass propagation and conservation. Protocorm-like bodies (PLBs) were induced from leaves in culture medium supplemented with plant growth regulators, and generated complete in vitro plants. To deepen on the mechanism underlying this morphogenetic route, this study is aimed to identify and characterize differentially expressed proteins during C. tigrina PLBs development. Leaves were inoculated in in culture medium Murashige and Skoog (MS) supplemented with 9 μM Thidiazuron, and PLBs t were collected after 30, 60 and 100 days in culture. Proteomic analysis performed by two-dimensional electrophoresis and MALDI-TOF mass spectrometry detected 122, 132 and 447 protein after 30, 60 and 100 days, respectively. During PLBs development, 36 proteins were differentially expressed. Proteins related to energy and carbohydrate metabolism, cell proliferation, protein processing, and secondary metabolism and phytohormones were assessed showing their relevance in the process of development of PLBs. Many proteins related to metabolic and energy processes were identified after 60 and 100 days in culture, suggesting their role in which is cell division and differentiation. Enolase was only present after 100 days in culture, thus being a candidate as a molecular marker in this developmental phase.
... The explants cultured on medium follows two regeneration pathways, organogenesis and somatic embryogenesis. In the somatic embriyogenesis pathway, bipolar structures regenerate from a single cell and they have no vascular connection [19]. Unlike somatic embryogenesis, unipolar structures occur in the organogenesis pathway and vascular connection is formed between the formed structures and the donor plant. ...
Primula is the largest genus belongs to Primulaceae family. The majority of the genus is comprising short-lived perennial herbaceous plants. Members of the genus have attractive flowers and are cultivated as bedding plants and flowering potted plants. Additionally, some of the species among the genus are economically important for ornamental plant industry. In commercial production systems, hybrids are especially propagated by seed, while primroses can also be propagated by dividing clumps. However, some problems such as irregular or late germination and low germination rate of the seeds in many primula species prevent mass propagation. Therefore, tissue culture techniques are valuable tools in breeding program, enlargement of genetic pool, gene transformation, as well as in conservation plant genetic materials in primula. In this review, common plant tissue culture techniques used in Primula species were briefly presented.
... In vitro somatic embryogenesis is an alternative method in achieving fast and large-scale plant propagation (Haccius, 1978). In somatic embryogenesis a plant or embryo is derived from a single cell. ...
... The formation of somatic embryos is strongly associated with the embryogenic competence of the explant cells. Possibly, the acquisition of embryogenic competence is related to the endogenous level of plant hormones, which favor tissue sensitivity to plant growth regulators present in the culture medium, which modulates events leading to the formation of the somatic embryo [21,28,29]. ...
... Yang sering terjadi adalah bahwa embrio somatik terbentuk pada permukaan kalus dan dengan mudah dapat dipisahkan dari selsel di sekelilingnya. Pada umumnya para pakar kultur jaringan menerima pendapat bahwa embrio somatik berasal dari satu sel dan tidak berhubungan langsung secara struktural dengan jaringan induknya (Haccius, 1978;Gray, 2000). Namun demikian, ada laporan penelitian yang menunjukkan bahwa embrio somatik dapat berasal dari sekumpulan sel. ...
Kelapa sawit (Elaeis guineensis Jacq.) merupakan penghasil minyak paling efisien di antara tanaman-tanaman penghasil minyak. Perbanyakan kelapa sawit biasanya dilakukan dengan biji, yang merupakan hasil persilangan antara kelapa sawit tipe dura dan pisifera. Hasil persilangan tersebut menghasilkan kelapa sawit tipe tenera. Tipe tenera inilah yang ditanam di kebanyakan perkebunan komersial. Oleh karena populasi tipe dura dan pisifera sebagai tetua adalah beragam secara genetik, maka populasi turunannya, yaitu tipe tenera, juga beragam, termasuk beragam dalam hal produktivitasnya: sebagian tinggi, sebagian rendah. Jika individu-individu kelapa sawit tipe tenera yang produktivitasnya tinggi diseleksi, lalu diperbanyak secara klonal, maka akan diperoleh populasi tanaman kelapa sawit tipe tenera yang produktivitasnya tinggi. Untuk kelapa sawit, sejauh ini perbanyakan klonal hanya bisa dilakukan dengan teknik kultur jaringan.
Buku yang ada di tangan pembaca ini memaparkan teknik tersebut, yaitu “Kultur Jaringan untuk Perbanyakan Klonal Kelapa Sawit”. Secara detil tatacaranya diuraikan dalam buku ini, termasuk teori yang mendasarinya dan sarana dan prasarana yang dibutuhkan. Mudah-mudahan buku ini bermanfaat di tengah-tengah langkanya publikasi ilmiah mengenai prosedur detil kultur jaringan kelapa sawit. Publikasi langka, kemungkinan besar karena tingginya nilai komersial teknologi ini.
... Embryos can be distinguished from adventitious shoots because they are bipolar, having both a shoot pole and a root pole, and do not have any vascular connections with the underlying parental tissues (18). Individual embryos were easily separated from the maternal tissues, as they were loosely attached at the radicle end to the mother explant (Fig. 1B). ...
A plant regeneration protocol was established from hypocotyl explants of in vitro grown seedlings of Asparagus officinalis and in vitro-proliferated shoots, respectively through either somatic embryogenesis or embryogenic callus. Types of auxins and cytokinins play an important role for producing somatic embryos. Friable calli with somatic embryos developed well in MS medium supplemented with 2.0-4.0 μM 6-benzylaminopurine (BAP) and 1.0-4.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 1-naphthaleneacetic acid (NAA) or indole-3-butyric acid (IBA). The highest 96.3% embryogenic calli and maximum number of 60.0 somatic embryos formation were obtained from each explant when the MS medium was fortified with 4.0 μM BAP and 2.0 μM 2,4-D. The best embryo germination occurred in 1.0 μM BAP supplemented ½ strength MS medium. The highest 95.2% of shoot proliferation was observed in embryogenic calli in MS medium containing 2.0 μM BAP and 1.0 μM IBA or NAA. In vitro-grown shoots were rooted in ½ strength MS medium with 0.5-4.0 μM IBA. Regenerants were transferred to vermicompost and successfully established under an ex vitro environment in garden soil with 75.0% survival rate.
... A multicellular origin proposed by Williams and Maheswarin (1986) to occur in some cases has not been unequivocally demonstrated. Haccius (1978) concluded that somatic embryos can derive from a single cell or proembryonal cell complexes which are derived from a single segmenting cell. The explants commonly used for investigation of SE in Arabidopsis are from immature zygotic embryos or the seedling SAM (Mordhorst et al., 1998;Gaj, 2001;Harding et al., 2003;Kurczyńska et al., 2007;Kadokura et al., 2018). ...
Medicago truncatula is now widely regarded as a legume model where there is an increasing range of genomic resources. Highly regenerable lines have been developed from the wild-type Jemalong cultivar, most likely due to epigenetic changes. These lines with high rates of somatic embryogenesis (SE) can be compared with wild-type where SE is rare. Much of the research has been with the high SE genotype Jemalong 2HA (2HA). SE can be induced from leaf tissue explants or isolated mesophyll protoplasts. In 2HA, the exogenous phytohormones 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) are central to SE. However, there are interactions with ethylene, abscisic acid (ABA), and gibberellic acid (GA) which produce maximum SE. In the main, somatic embryos are derived from dedifferentiated cells, undergo organellar changes, and produce stem-like cells. There is evidence that the SE is induced as a result of a stress and hormone interaction and this is discussed. In M. truncatula, there are connections between stress and specific up-regulated genes and specific hormones and up-regulated genes during the SE induction phase. Some of the transcription factors have been knocked down using RNAi to show they are critical for SE induction (MtWUSCHEL, MtSERF1). SE research in M. truncatula has utilized high throughput transcriptomic and proteomic studies and the more detailed investigation of some individual genes. In this review, these studies are integrated to suggest a framework and timeline for some of the key events of SE induction in M. truncatula.
... Confirmation of somatic embryogenesis is based on histological evidence that the structure is bipolar and has no vascular connection to the explant [43]. The most suitable regeneration systems for transformation are direct or repetitive production of somatic embryos or de novo shoot organogenesis, which originate from single cells of the epidermal layer [44,45]. ...
Nodal segments of sandalwood were cultured on MS medium amended with different plant growth regulators in varying concentrations to search out higher in vitro response leading to plantlet regeneration via somatic embryogenesis and/or organogenesis. Higher proportion of direct somatic embryogenesis, number(s) of somatic embryo per explant and plantlet regeneration via direct organogenesis were recorded on MS medium supplemented with a moderate concentration of TDZ (1.0 mgl-1) in combination with comparatively a lower concentration of NAA (0.5 mgl-1). A relative higher concentration of BAP (1.0-2.0 mgl-1) in combination with a lower concentration of NAA (0.5 mgl-1) promoted frequency of indirect somatic embryogenesis. Ratio of organ formation directly from surface of cultured explants was recovered from culture medium fortified with a higher concentration of BA at the concentration of 4.0 mgl-1 in combination with a lower concentration of NAA (0.5 mgl-1). Maximum plantlets regenerated via somatic embryogenesis (direct and/or indirect) on regeneration medium supplemented with 2.0 mgl-1TDZ in combination with 1.0 mg l-1GA3, while plantlets in higher frequencies via indirect organogenesis was attained with regeneration medium amended with comparatively lower concentration of TDZ (1.0 mg l-1) in combination with 0.5 mgl-1 GA3 and 0.5 mgl-1 NAA. The plantlets were transferred to pots and hardened in Environmental Growth Cabinet and Net House during preliminary weaning period and transferred to field successfully. Morphologically normal plants were recovered.
... In some samples, also, the shoot apical meristem became larger, which suggests its possible participation in callus production. De novo formation of shoots appeared on regeneration media, as no structures growing on the callus met the criteria for somatic embryogenesis (Haccius 1978). The best response of explants (79-80%) was noted for control medium (C) and medium with honey (SH), followed by PVP and CH medium (Table 4). ...
Sorghum bicolor is a recalcitrant species for tissue culture regeneration and genetic transformation. Browning of explants is one of the factors limiting organ and tissue cultures. To overcome this, callus tissue was initiated from the shoot tips of in vitro germinating seeds (S. bicolor cv. Róna 1), and then cultured on modified MS media (Murashige and Skoog in Physiol Plant 15:473–497, 1962). In the first experiment, we tested callus induction on several media supplemented with casein hydrolysate, polyvinylpyrrolidone, honey, and sucrose. The best callus induction was recorded for the medium with honey and sucrose (80.0%) and for control medium (79.8%). Shoot regeneration was tested on the MS medium with 6-benzylaminopurine (BAP) supplemented with honey and sucrose at a 1:1 ratio (by weight) or with sucrose only. The highest percentage of calluses regenerating shoots was noted for those induced on the medium with sucrose and honey—approx. four times higher when compared to the control. Rooted plantlets were acclimatized with a 92% survival rate. In the second experiment, we analyzed culture responses to various ways of honey application to the induction media: honey (autoclaved or filtered) in presence or absence of sucrose. Supplementation of the medium with fructose, glucose, and maltose at a proportion typical for honey was also investigated. The explant and callus survival rates were similar to those of the honey–sucrose combination in the first experiment. Only presence of both sucrose and honey in the induction medium improved the total regeneration rate to 37.9% over the control (18.8%). Sucrose and honey appear to act synergistically for shoot regeneration in callus cultures of sorghum.
... The increased N/C ratio observed in the embryogenic areas (ranging from 4.5% for subepidermal cells in the original cotyledons to 42.6% for embryogenic cells) may have reflected the degree of dedifferentiation to reach the embryogenic state, in contrast to the increased vacuolation that reflects advancing cell differentiation and loss of competence (Margara, 1982). Few-celled proembryos, multicellular embryogenic areas (possibly the proembryonal cell complex mentioned by Haccius (1978), and globular-stage embryos could be distinguished by day 21 (Figure 2A,B). Subsequently, heart-shaped and torpedo-shaped embryos were formed, and by day 28, many embryos had reached the cotyledonary stage, with shoot and root meristems and differentiation of a closed vascular system ( Figure 2C). ...
Hardwood species are valuable biological resources that have an important role in the economy and ecology of ecosystems worldwide. Non-zygotic or somatic embryogenesis (SE) is a powerful tool in plant biotechnology as it is a form of clonal propagation, amenable to cryopreservation of valuable germplasm and genetic transformation including gene editing. The SE process involves five steps and includes somatic embryo induction, proliferation, maturation, plantlet conversion, and subsequent plant acclimatization. This review aims to provide a general overview of these steps in different SE systems developed for hardwood species. Factors that influence the induction stage such as the age of the donor plant, genotype and culture media are discussed. The role of different explant types, i.e. zygotic embryos and non-zygotic tissues, such as roots, flower tissues, nodes, internodes, leaves or shoot apices, in SE induction are especially emphasized. Histological studies of the origin of somatic embryos and the sequence of events leading to their development from initial explants are assessed. Maintenance of embryogenic capacity carried out by subculture of embryogenic inocula on semisolid or liquid media through cell suspension cultures or by temporary immersion systems is described. At present, the main concerns associated with the application of SE for large-scale propagation of elite hardwoods are related to the embryo maturation, germination, and plantlet conversion steps, and these are highlighted in this review. Finally, molecular aspects associated with somatic embryo induction and development are also described. Attempts to overcome the hurdles identified in the embryogenic process, and future lines of research are proposed.
... Chrysanthemum micropropagation involves in vitro cuttings with existing meristems (axillary or apex), while breeding -adventitious organogenesis or somatic embryogenesis. The latter is a biological process with somatic embryos being formed from vegetative cells [Haccius 1978]. It is commonly regarded as one of the most effective micropropagation methods, facilitating the generation of a large number of plants in a very short time [Kulpa 2012]. ...
The objective of the study has been to determine the usefulness of six different ISSR (Inter Simple Sequence Repeat) primers for the genetic stability in the chrysanthemum plants produced through somatic embryogenesis (SE). The research material consisted of the lines of two cultivars: ‘Lady Salmon’ (LS1-LS5) and ‘Lady Vitroflora’ (LV1-LV5). The plants derived from meristems constituted the control. The primers used in the PCR reaction have shown a notable polymorphism between the genotypes analyzed. All the primers (S1-S6) demonstrated a polymorphism in the inter-microsatellite region; the highest polymorphism in ‘Lady Salmon’ and ‘Lady Vitroflora’ lines with primer S3, whereas the lowest – in 'Lady Salmon' cultivar with primers S5 and S6, and in ‘Lady Vitroflora’ – with primer S1. UPGMA dendrogram clearly separated all the cultivars and its lines into two main clusters and two subclusters. ISSR markers can be applied to assess the genetic stability of SE-derived chrysanthemum.
... The formation of embryos from the vegetative tissues can also be induced under in vitro condition. A somatic embryo (SE) is a bipolar structure that has no vascular connection with the maternal tissue of the explant [3]. The process of somatic embryogenesis (SEG) could vary depending on if SE developed directly from explant or from an intervening callus phase. ...
Somatic embryogenesis (SEG) is one of the best techniques for mass production of economically important plants. It is also used for the study of morphology, anatomy, physiology, genetics and molecular mechanism of embryo development. Somatic Embryos (SE) are bipolar structures that develop from a cell other than a gamete or zygote. SEG reflects the unique developmental potential of plant somatic cells, resulting in the transition of the differentiated somatic cells to embryogenic cells to follow the zygotic embryo stages. There are several biochemical and physiological processes that transformed a single somatic cell to a whole plant. SE studies provide insight into cell mechanisms governing the totipotency process in plants. Previously, in vitro studies have suggested the role of various regulatory genes in embryogenic transition that are triggered by plant hormones in response to stress. The omic studies identify the specific genes, transcripts, and proteins required for somatic embryogenesis development. MicroRNAs (miRNAs) are small, 19–24 nucleotides (nt), non-coding small RNA regulatory molecules controlling a large number of biological processes. In addition to their role in SEG, miRNAs play vital role in plant development, secondary metabolite synthesis and metabolism of macromolecules, hormone signal transduction, and tolerance of plants to biotic and abiotic stresses. During last decade several types of miRNAs involved in SEG have been reported. Among these miRNAs, miR156, miR162, miR166a, miR167, miR168, miR171a/b, miR171c, miR393, miR397 and miR398 played very active role during various stages of SEG. In this review, we highlighted the role of these as well as other miRNAs in some economically important plants.
... Chrysanthemum micropropagation involves in vitro cuttings with existing meristems (axillary or apex), while breeding -adventitious organogenesis or somatic embryogenesis. The latter is a biological process with somatic embryos being formed from vegetative cells [Haccius 1978]. It is commonly regarded as one of the most effective micropropagation methods, facilitating the generation of a large number of plants in a very short time [Kulpa 2012]. ...
Chrysanthemum (Chrysanthemum × grandiflorum Ramat./Kitam.) occupies a high position on the gardening market, and its production continues to increase every year. To satisfy the growing needs of the market, new methods of propagation and breeding are being sought. One of the most efficient regenerative methods is somatic embryogenesis (SE), which can be used both for micropropagation of the plants and for breeding of new cultivars at the stage of regeneration from singular somatic cells. The greatest SE advantage is its efficiency, specifically a high number of somatic embryos regenerated from a single explant. An underrated but very important aspect of somatic embryogenesis is a possible variation in plants regenerated applying that method, often resulting from adding high concentrations of growth regulators to the medium at the ES induction stage, and it is related to the callus phase which frequently mediates the regeneration of somatic embryos. The variation can be applied in new cultivars breeding programmes. To detect the variation at the genetic level, the molecular markers are used. Inter Simple Sequence Repeat primers (ISSR) based on the amplification of DNA segments are contained between two opposite-directed repeat regions which comprise microsatellite repeat sequences. These repetitions are characterized by a high variation and they are widespread throughout the genome.
The objective of the study has been to determine the usefulness of six different ISSR primers for the genetic stability research in the chrysanthemum plants obtained through somatic embryogenesis. The research material consisted of the lines of two cultivars: ‘Lady Salmon’ (LS1-LS5) and ‘Lady Vitroflora’ (LV1-LV5). The plants obtained from meristems constituted the control. DNA was isolated from the chrysanthemum leaves with the use of Genomic Mini AX Plant kit (A&A Biotechnology firm). The electrophoretic separation was performed on 1.5% agarose gel with the voltage current equaling 90 V for 20 minutes and 110 V for the next 90 minutes. The analysis of the reaction products was made using GelAnalyzer 2010 program. The primers used in the PCR reaction have shown a notable polymorphism between the genotypes analyzed. In ‘Lady Salmon’ cultivars 236 products were obtained; 23 bands were polymorphic, while in ‘Lady Vitroflora’ 221 products were produced, from which 19 showed a variability. All the primers (S1-S6) showed a polymorphism in the inter-microsatellite region; the highest polymorphism in 'Lady Salmon' and 'Lady Vitroflora' lines with primer S3, whereas the lowest – in 'Lady Salmon' cultivar with primers S5 and S6, and in 'Lady Vitroflora' cultivar with primer S1. The highest average polymorphism was shown with primer S2 and the lowest – with primer S5, which allowed for selecting the primers to be used in genetic variation analyzes. ISSR markers find their application in the assessment of genetic stability of Chrysanthemum × grandiflorum Ramat./Kitam. obtained as a result of somatic embryogenesis.
... SE is a sexual-independent development process that yields a bipolar embryo derived directly from somatic tissue through a zygotic-embryogenesis resembling process [16]. SE is able to give rise to the cellular totipotency in higher plants as well has the advantage over organogenesis of true type regeneration and normal seedling development and is able to begin from a single cell [17] or small groups of cells [18]. However, up today whether SE begins from a single cell is an open question to be answered. ...
Common bean Phaseolus vulgaris L. has been shown to be a recalcitrant plant to induce somatic embryogenesis (SE) under in vitro conditions. An alternative strategy to yield SE is based upon the use of a cytokinin (benzyladenine) coupled with osmotic stress adaptation instead of the auxin-inducing SE in common bean. Here we described the induction of proembryogenic masses (PEM) derived from the apical meristem and cotyledonary zone of zygotic embryos, from which secondary SE indirect embryogenesis emerged. Maturation of SE was achieved by using osmotic stress medium and converted to plants. Long-term recurrent SE was demonstrated by propagation of PEM at early stages of SE. This protocol is currently being applied for stable genetic transformation by means of Agrobacterium tumefaciens and biobalistics as well as basic biochemical and molecular biology research.
... Inspite of long history of isolated plant cell, tissue, organ and embryo culture methods in vitro, as well as abundance of biochemical, physiological data and some other aspects, characteristics of morphogenetic transformations from the very first stages have been insufficiently studied. Haccius contributed to the study of morphogenetic processes in vivo and in vitro significantly (Haccius 1965(Haccius , 1973(Haccius , 1978Haccius & Bhandari 1975;Haccius & Lakshmanan 1969). Her papers deserve special attention. ...
Morphological processes in explants of leaves, axes and shoots of lilies, hyacinth, daffodil
and gladiolus during regeneration in vitro undergo gemmorhizogenesis. Shoot formation precedes the
differentiation of adventive roots. Practically all living tissues of explants including the parenchyma
sheath of vascular bundles are involved in the formation of meristematic clumps. As a rule epidermis
doesn’t take part in this process immediately. It is due to its much earlier and deeper specialization.
During regeneration, the compound branched hydrocyte system differentiates in explants of different
morphological nature. It serves redistribution of inner resources within explants and resources of the
culture medium as well which is necessary for the differentiation of de novo shoots and adventitious
root rudiments. Differences in morphogenetic reactions in vitro of explants of different morphological
nature are non-significant and they depend only to a less degree on its taxonomical affiliation.
... These structures were similar to those previously reported in Panicum maximum [32,33], Paspalum notatum [34,24]. Additionally, the recovery of chemical-or radiation-induced mutations or genetically transformed plants should be possible using this technique since somatic embryos are generally believed to arise from single cell [35]. And then, the calli with white color were transferred onto the same medium, and after two weeks of the culture, the somatic embryos with green color were differentiated (Fig. 4F). ...
... Another point of discussion is a single-or multiple-cell origin of somatic embryoids. Induction of somatic embryo from a superficial cell possibly indicates its unicellular origin (Haccius 1978) or from subepidermal cells, representing a multicellular origin (Tisserat et al. 1978). The various events occuring during somatic embryogenesis have been schematically represented in Fig. 1.3. ...
In 1902 Gottlieb Haberlandt proposed the idea to culture individual plant cells on artificial nutrient medium. Although he failed to culture them due to poor choice of experimental materials and inadequate nutrient supply, he made several valuable predictions about the nutrients’ requirement for in vitro culture conditions, which could possibly induce cell division, proliferation and embryo induction. Tissue culture has now become a well-established technique for culturing and studying the physiological behaviour of isolated plant organs, tissues, cells, protoplasts and even cell organelles under precisely controlled physical and chemical conditions. Micropropagation is one of the most important applications of plant tissue culture. It provides numerous advantages over conventional propagation like mass production of true-to-type and disease-free plants of elite species in highly speedy manner irrespective of the season requiring smaller space and tissue source. Therefore, it provides a reliable technique for in vitro conservation of various rare, endangered and threatened germplasm. Micropropagation protocols have been standardized for commercial production of many important medicinal and horticultural crops. Somatic embryogenesis is an extremely important aspect of plant tissue culture, occurring in vitro either indirectly from callus, suspension or protoplast culture or directly from the cell(s) of an organized structure. Advantages of somatic embryogenesis over organogenesis include several practical means of micropropagation. It reduces the necessity of timely and costly manipulations of individual explants as compared to organogenesis.
... All of the previous research dealing with strawberry somatic embryogenesis included only limited histological examination of the obtained structures which were classified as somatic embryos. However somatic embryos are defined as bipolar structures, which do not have a vascular connection with the mother tissue at any time of their life 16 and develop through characteristic embryological stages 17 . None of the above mentioned reports dealing with somatic embryogenesis in strawberry presented unambiguous histological proof of somatic embryogenesis. ...
This study was conducted to examine the possibility of inducing somatic embryogenesis in strawberry using tissue culture technique. Experimental works included evaluation of five published protocols which did not prove the induction of somatic embryogenesis by histological means, in addition to examine, alternative approaches. The embryogenic nature of the regenerating tissues was checked by histological analysis. Factors regarding the plant materials (cultivars and explant source) and those concernal with external effects (medium component, light and temperature) were studied.
Results of repeating published protocols indicated the formation of callus and shoot-like structures (protocol of Wang et al, 1984), or somatic embryo-like structures (SELS) and shoot buds (protocols of Donnoli et al, 2001 and Biswas et al,2007). Protocol (4) of Husaini et al(2008) also produced callus, SELS or direct shoot regeneration, while protocol 5 (Kordestani and Karami, 2008) resulted in only callus. Morphohistological examination did not prove the induction of somatic embryo in the five tested protocols. The process of somatic embryogenesis in strawberry was further studied utilizing different combinations of plant growth regulators, explants sources and cultivars. Structures resembling somatic embryos were formed at high frequencies from petiole segments on medium amended with 4.0mg/l 2,4-D+ 0.5mg/l BA + 50 g/l sucrose (Exp1), or medium with 1.5 mg/l TDZ +4.0 mg/l Picloram (Exp. 3), while medium with several combinations OF BA + glutamin only formed callus (Exp.2). However, histological studies on the regenerated structures did not confirm their embryogenic nature, indicating that strawberry could be recalcitrant in term of somatic embryogenesis
... Furthermore, a non-T-shaped four-cell proembryo was also observed. After disordered cell division, this proembryo developed into an embryonic clump (Fig. 4), which was referred to by Haccius (1978) as a proembryonal cell complex, from which one or multiple SEs were subsequently developed. Similar reports have been published on related histological observations of D. caespitosa (Trigiano et al., 1989;Vasilenko et al., 2000) and Coffea arabica (Quiroz-Figueroa et al., 2002). ...
In this study, a procedure for the histological observation of the somatic embryogenesis in Dieffenbachia 'Anna' was developed. Thin slices of secondary somatic embryos (SSEs) were used as explants. Somatic embryogenesis tended to be derived from a single cell. Upon further differentiation, the single cell became an embryogenic cell mass, and somatic embryos (SEs) subsequently formed under the influence of 3, 6-dichloro-2-methoxybenzoic acid (dicamba) and l-phenyl-3-(1, 2, 3-thiadiazol-5-yl) urea (TDZ). The cytological origin and early proembryoid differentiation process were initiated after 6 days of culture. A single embryogenic cell initiated asymmetric cell division to form an apical cell and basal cell. A two-celled proembryo subsequently developed from the basal cell, forming a three-celled proembryo. After 9 days of culture, the apical cell underwent anticlinal division, and prior the two cells of periclinal division to form a T-shaped four-celled proembryo. After 12 days of culture, the cells continued to divide and formed an eight-celled proembryo. After 15-18 days of culture, 8-10 cells formed a rudimentary proembryo, revealing the primary structure of the embryo. After 24 days of culture, a 16-celled proembryo was formed. The multicellular embryoid had formed within the explants, which gradually emerged to the surface of the explants. Each embryoid exhibited a thick cell wall, isolated with the surrounding cells from the embryonic base of the suspensor construction. After 27 days of culture, the proembryo emerged from the explant surrounding, and the early globular embryoid with protoderm. After 12 weeks of culture, the explants in the original medium formed embryoid clusters with various developments were visible. Histological observation showed that the globular embryos separated from the explants, with distinct protoderm and Y-shape closed pro-cambium. During the development of the somatic embryos, an asynchronous phenomenon was observed in the mature embryo stage of the scutellum and coleoptilar embryos, but not when the mature somatic embryos converted to emblings.
... Embryos are defined, with general acceptance (Brown et al., 1995), as new individuals that develop both a shoot apex and a discrete radicular end (Haccius 1978). Kumar et al. (2003) and reported on embryogenesis and plant regeneration in cultured anthers of cucumber (cvs. ...
This book is a comprehensive review of secondary metabolite production from plant tissue culture. The editors have compiled 12 meticulously organized chapters that provide the relevant theoretical and practical frameworks in this subject using empirical research findings. The goal of the book is to explain the rationale behind in vitro production of secondary metabolites from some important medicinal plants. Biotechnological strategies like metabolic engineering and the biosynthesis, transport and modulation of important secondary metabolites are explained along with research studies on specific plants. In addition to the benefits of secondary metabolites, the book also aims to highlight the commercial value of medicinal plants for pharmaceutical and healthcare ventures.
Topics covered in this part include:
1. In vitro propagation and tissue culture for several plants including Withania somnifera (L.) Dunal, Aloe vera, Oroxylum indicum (L) Kurz, Ocimum basilicum L, Rhubarb, Tea, and many others (including plants in Northern India).
2. Genetic Improvement of Pelargonium
3. Bioactive Components in Senna alata L. Roxb
4. Plant tissue culture techniques
The book caters to a wide readership. It primarily prepares graduate students, researchers, biotechnologists, giving them a grasp of the key methodologies in the secondary metabolite production. It is a secondary reference for support executives, industry professionals, and policymakers at corporate and government levels to understand the importance of plant tissue culture and maximizing its impact in the herbal industry.
La información presentada en cada una las prácticas del presente manual son del área microbiana, vegetal y animal, y sólo tiene el objetivo de servir como guía de apoyo para practicar en el laboratorio; los materiales y métodos descritos puede variar durante el desarrollo de la práctica, y por lo tanto, en los reportes se deberá incluir aquellos cambios o procesos adicionales realizados durante la práctica.
Ya que esta publicación está destinada a la docencia, se pide que el contenido de un reporte de práctica debe contener los siguientes apartados:
1. Título y número de práctica
2. Nombre completo del alumno (anexe grupo, calendario, correo electrónico y fecha de entrega)
3. Introducción (con apoyo en revisión de literatura)
4. Objetivo (s)
5. Materiales y métodos realizados durante la práctica
6. Resultados y Discusión (con apoyo en revisión de literatura)
7. Conclusiones
8. Literatura consultada
Los cuadros y figuras deberán llevar leyenda y estar citados en el texto con numeración progresiva. Asimismo es conveniente mencionar que el objetivo puede formar parte de la introducción. Recuerde que la discusión se hace con base en los resultados obtenidos y la revisión de literatura. Las conclusiones es la única sección que puede ir puntualizada o numerada. Por su parte la literatura citada será aquella que se utilizó para documentar el reporte, apegada a formato científico, para lo cual es necesario revisar artículos en revistas especializadas, libros técnicos y sitios en internet adecuados.
Los reportes son personales, aun cuando alguna pr ctica se haya llevado a cabo en equipo. Se entregarán directamente al profesor, en formato digital en procesador de palabras compatible, de acuerdo a la programación indicada en clase.
Cualquier asunto no contemplado en la presente guía de prácticas, se deberá tratar directamente con el profesor.
Tilia species are valuable woody species due to their beautiful shape and role as honey trees. Somatic embryogenesis can be an alternative method for mass propagation of T. amurensis. However, the molecular mechanisms of T. amurensis somatic embryogenesis are yet to be known. Here, we conducted comparative transcriptional analysis during somatic embryogenesis of T. amurensis. RNA-Seq identified 1505 differentially expressed genes, including developmental regulatory genes. Auxin related genes such as YUC, AUX/IAA and ARF and signal transduction pathway related genes including LEA and SERK were differentially regulated during somatic embryogenesis. Also, B3 domain family (LEC2, FUS3), VAL and PKL, the regulatory transcription factors, were differentially expressed by somatic embryo developmental stages. Our results could provide plausible pathway of signaling somatic embryogenesis of T. amurensis, and serve an important resource for further studies in direct somatic embryogenesis in woody plants.
The ultrastructure, morphology, and histology of somatic embryogenesis in pearl millet (Pennisetum glaucum) were examined using light and electron microscopic techniques. Somatic embryogenesis was initiated from zygotic embryo explants cultured 8 d after pollination. Formation of a ridge of tissue began 3–4 d after culture (DAC) by divisions in the epidermal and subepidermal cells of the scutellum. Ridge formation was accompanied by a decrease in vacuoles, lipid bodies, and cell size, and an increase in endoplasmic reticulum (ER). Proembryonic cell masses (proembryoids) formed from the scutellar ridge by 10 DAC. Proembryoid cells had abundant Golgi bodies and ER while the amounts of lipids and starch varied. Somatic embryos developed from the proembryonic masses 13 DAC and by 21 DAC had all the parts of mature zygotic embryos. Although shoot and root primordia of somatic embryos were always less differentiated than those of zygotic embryos, scutellar cells of somatic and zygotic embryos had similar amounts of lipids, vacuoles, and starch. Somatic scutellar epidermal cells were more vacuolated than their zygotic counterparts. In contrast, somatic scutellar nodal cells were smaller and not as vacuolated as in zygotic embryos. Somatic embryogenesis was characterized by three phases of cell development: first, scutellar cell dedifferentiation with a reduction in lipids and cell and vacuole size; second, proembryoid formation with high levels of ER; and third, the development of somatic embryos that were functionally and morphologically similar to zygotic embryos.
Immature embryos as well as explants obtained from young inflorescences of Pennisetum americanum (pearl millet) give rise to callus tissues on nutrient media containing 2,4-dichlorophenoxyacetic acid (2,4-D). A compact and pale-yellow callus that arises from the peripheral cells of the scutellum, and from the young inflorescences, undergoes further organized growth. When transferred to a 2,4-D-free medium, supplemented with indole-acetic acid or kinetin, or both, embryoids are formed in the organized areas of the callus. Embryoids show a bipolar organization with a shoot-coleorhiza (root) axis and have a coleoptile-like structure surrounded at the base by a cup-shaped structure that resembles the scutellum in texture and morphology. Embryoids show bilateral or radial symmetry and “germinate” in vitro to form plants that have been grown to maturity in soil. Similar embryogenic callus cultures have been produced from young inflorescence tissues of hybrid Pennisetum, a triploid sexually sterile hybrid of P. americanum x P. purpureum. Plants derived from these have also been transferred to soil. The regenerated plants showed normal chromosome numbers.
Embryogenic cell lines of Vitis rotundifolia were produced from immature zygotic embryo explants obtained by culturing ovules, harvested at 20 d postanthesis, for 8 wk and then dissecting embryos from them. Ovules cultured on Nitsch and Nitsch medium with naphthoxyacetic acid and benzyladenine (BA) produced a brown exudate, necessitating three transfers to fresh medium at 2-wk intervals during the 8-wk culture cycle. Zygotic embryos that were subsequently isolated from cultured ovules and placed on the same medium produced a heterogenous callus from which eventually emerged embryogenic cell lines. A higher percentage of ovules from cultivars ‘Dixie’, ‘Fry’, ‘Nesbitt’, and ‘Welder’ produced zygotic embryos (31%–39%) than did those from ‘Carlos’ (3%). A higher percentage of ‘Fry’ ovules produced embryogenic lines from cultured zygotic embryos (6.3%) than did those of the other four cultivars (1%–1.6%). Embryogenic cell lines were white and composed of variably sized cell clusters, somatic embryos, and embryonic tissue embedded in a watery matrix. These lines were maintained for over 1 yr on modified Murashige and Skoog (MS) medium lacking growth regulators by transfer of selected cell clusters every 6 wk. White, opaque somatic embryos grew directly from cell clusters and passed through recognizable developmental stages. Germination was induced by transfer of somatic embryos to MS medium with BA. Although 80%–100% of embryos germinated, plant recovery was low due to poor shoot development.
In Hevea, currently described culture conditions allow the induction of two modes of embryogenesis in callus formed on excised portions of the internal integument of immature seeds. One mode is of unicellular origin and is transitory, only resulting in the production of globular proembryos. The other is of multicellular origin and produces embryos. Specific culture conditions appear to favor one or the other mode of development in a given genotype. The ontogenesis of embryos of multicellular origin bypasses the classical stages of zygotic embryogenesis. The structural abnormalities observed in most somatic embryos are probably responsible for the low germination rates obtained.
Callus initiation of sections of megagametophytes of Larix decidua occurs just below the cut surface and is followed by the formation of one or more long protruding cells. The long cells then divide transversely at their tips to yield small cytoplasmically-dense terminal cells. The latter divide, forming loose aggregates of dense cells, microcalli, which develop long cells that radiate in all directions and divide terminally to produce aggregates of small cells. This long/short cell alternation is repeated a few times. Eventually the aggregates divide in a polarized manner producing files of long cells predominantly in one direction. These loosely bundled long cells form a suspensor-like structure. The meristematic small cells continue dividing forming a mass of embryonal cells. This early embryoid eventually turns green and produces both a root and shoot. Haploid embryoids are also derived from long cells in which karyokinesis but not cytokinesis occurs, resulting in a four-nucleate coenocyte. The nuclei migrate to one pole and become surrounded by cell walls. The cells thus formed are the originators of a new embryoid.
Callus tissue cultures were initiated from immature embryos, mature embryos and young inflorescences of Guinea grass (Panicum maximum Jacq.) on Murashige and Skoog's (MS) medium supplemented with 2.5–10 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). Calluses were transferred onto the same nutrient medium with 0.2 mg/l 2,4-D, or without 2,4-D. In callus cultures derived from immature embryos and young inflorescence segments, plantlets were produced via somatic embryogenesis after 3–5 wk. Young plants were successfully transplanted to pots and grown in the greenhouse. Plant development in callus obtained from mature embryos took place through the organization of shoot meristems. Regenerated plants were shown to have the normal tetraploid chromosome number of 2n = 4x = 32.
Se presentan 11 actividades de laboratorio como guía para alcanzar a cubrir los tres procesos de micropropagación vegetal de una manera práctica.
In the present study we examined the possibility of propagating different Cyclamen species (C. africanum Boiss. and Reut., C. cilicium Boiss. and Heldr., C. coum Mill., C. hederifolium Ait., C. persicum Mill., C. purpurascens Mill.) including some of their subspecies and cultivars in vitro using explants of adult plants. For this purpose two protocols have been applied to eleven genotypes combined with mostly four explant types (placentas with ovules, leaves, petioles and peduncles). The use of these protocols has given rise to either somatic embryo-like structures and/or adventitious shoots in all genotypes. This way it was possible to propagate each of the examined genotypes in vitro using explants of adult plants in a time less than one year. These results may be used in breeding and propagation of Cyclamen as an ornamental plant and as a medicinal plant.
Bananas and plantains are the most important staple food crop in the world. The production of banana is hampered by various stresses. Genetic transformation has become an important potential tool in developing improved banana with desired agronomic traits, which is highly difficult to achieve through conventional breeding. To be successful, genetic engineering technique requires reliable, efficient in vitro regeneration protocol through tissue culture. Plant regeneration through somatic embryogenesis has become an important tool due to high proliferation potential, minimal genetic instability, and single-cell origin which in turn reduces the formation of chimera. However, banana is highly recalcitrant toward the development of somatic embryogenesis. Considerable progress has been achieved in the regeneration of banana through somatic embryogenesis, but there are still many factors to overcome. In present review key factors such as age of the explant, genotype, and plant growth regulators affecting the induction and regeneration of plants by embryogenic callus are discussed. This review also provides special focus on methods being applied in plant transformation through somatic embryogenesis, different factors which affect somatic embryogenesis, and various strategies to improve the transformation efficiency using somatic embryogenesis.
The genus Dactylis comprises a small and systematically rather isolated group in the tribe Festuceae of the grass family (Stebbins and Zohary 1959). The best known and most economically important species is Dactylis glomerata L., commonly referred to as “orchardgrass” in the United States and as “cocksfoot” in Europe, especially in the British Isles. It is a cool-season forage grass grown for both pasture and hay. Although it is a native of Europe, it has existed in North America for more than 200 years (Jung and Baker 1985).
Freesia is a small genus in Iridaceae, comprising 11 species. The common freesia (Freesia refracta Klatt.) was originally found in southern Africa (Goldblatt, 1982), but is now distributed in many parts of the world. The plant, normally 25–45 cm in height, has branched stems, slender leaves, and bulb-like tunicated corms. The flower possesses three stamens and an inferior ovary with three carpels. A normal plant has two types of roots: the thick contractile roots function mainly to support the plant, whereas the numerous thin roots transport materials from the soil to the plant (Ruzin, 1979). Freesia refracta can be propagated either sexually through seeds or vegetatively through corms. The seeds of freesia are difficult to obtain, and it usually takes years for seeds to develop into mature plants. Therefore, corms are practically used to propagate plants.
Big bluestem (Andropogon gerardii Vitman), little bluestem (Schizachyrium scoparium (Michx.) Nash), and Indiangrass (Sorghastrum nutans (L.) Nash) are important warm-season, perennial grasses native to North America (Weaver 1954). They are widely distributed throughout the eastern two thirds of the United States, and are major species of the tall-grass region of the central United States. Tall-grass prairie once occupied about 3% of the North American continent but now is found in only isolated remnants (Knapp and Seastedt 1986), since the most productive areas have been converted to cropland (Voigt and MacLauchlan 1985)
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