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PLANT BIOTECHNOLOGY

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PLANT BIOTECHNOLOGY

 

Ayesha Tungekar 

 

 

 

SOMATIC EMBRYOGENESIS

 

INTRODUCTION:

Somatic embryogenesis is the initiation and development of an embryo or plant from a single cell or group of vegetative cells in in-vitro conditions. In somatic embryogenesis, seeds are produced without endosperm and outer coat. First, a callus is formed followed by the development of embryos.

 

Events of somatic embryogenesis

Image: Events of somatic embryogenesis

Source: https://biocyclopedia.com/index/biotechnology/plant_biotechnology/

HISTORY OF SOMATIC EMBRYOGENISIS

In the year 1950s F.C Steward demonstrated somatic embryogenesis from free embryos. He is considered one of the pioneers of plant tissue cultures. Steward along with his co-workers working at Cornell University was the first to use coconut water for culturing of Daucus carota(carrot) explants. These explants showed vigorous proliferation of the Dacus carota explants. Later by using solidified coconut milk agar medium Steward and his co-worker’s cultured cell suspension of carrot callus obtained from carrot embryos. It was observed that numerous embryos developed from cells of callus tissue. This finding opened a new gateway for micropropagation of plants at a high and faster rate and also achieving totipotency of plant cell.

Harry Waris also made a remarkable contribution in the field of plant tissue culture. He cultured Oenanthe aquatica in a medium containing glycine for about 3-4 months. Initially, plants appeared normal but after some time they became morbid and growth halted. However,

 

surprisingly later on it was seen that new plants were generated from root tips. Later, these new plants called neomorphs were reproduced from groups of cells detached from the colorless outgrowth of the leaf epidermis. From the experiment, it was observed that the group of cells originating from roots, epidermis developed into embryos.

 

Pathway of Somatic Embryogenesis: Direct and Indirect Pathway

 

Direct Somatic Embryogenesis

In this, there is no callus formation and the embryos are derived from either a single cell or a group of cells. The explant used is zygotic embryos and it requires less nurture as they already have an embryonic property. The exception to these is the carrot (Daucus carota) and alfalfa. The cell division observed in direct somatic embryogenesis is more compact. Examples of direct somatic embryogenesis are immature embryos or cotyledons from Prunus persica(peach), cherry.

 

Indirect Somatic embryogenesis:

Here the explants form callus first and then embryo. As there is callus formation different degrees of compactness is seen.  On supplementation of embryonic callus with 2,4Dichlorophenoxyacetic acid, 6-BAP(Benzyl amino purine) they form Pre-embryogenic callus on or within the surface of callus from where the embryos originate from a single or cluster of cells. Example: young stem of  Euphorbia pulcherrima (Poinsettia), leaves of Vitis vinifera(grapes), embryos of Mangifera indica(mango).

 

Different stages of somatic embryogenesis:

 

Phase 0: Initiation

In this phase, the cell clusters acquire competence for the development of embryos. The cell clusters acquire competence under influence of embryos.

Phase1: Proliferation

The proliferation of embryogenic cell clusters is placed in a medium that is auxin-free.

Phase 2: Maturation:

It involves the shape attained by the growing embryos. Globular stage, heart stage, torpedo stage, cotyledonary stage are seen in dicots whereas in monocots globular, scutellar, coleptillar are seen in monocots.

 

GENETICS OF S.E

An important factor affecting S.E is the genotype of the plant. A lot of genotypic variation exists between the genus of the same species. Plant development and its physiology affect S.E. Tissue exhibiting a high rate of metabolic activity and low level of differentiation will promote. For example, the Shoot apex of Clematis florida (Clematis multi-blue) are easier to induce S.E among young stems and leaves. Similarly, it is easier to induce somatic embryogenesis in leaves of Anthuriuln andraeanum than in the other explants. The Leafy cotyledon2 and Baby boom are two marker genes seen after 5-6 days after the initiation of culture. Chromatin state of LAFL Gene which determines the pathway of somatic embryogenesis i.e. direct or indirect.

LAFL network consists of LEC-1, LEC,-2. LEC1encodes NF-YB9 (Nuclear factor protein), FUS3 and ABI.LAFL is the central regulatory network that plays role in embryo identity and seed maturation and the LAFL genes by interacting with upstream and downstream genes plays a role in regulating different aspects of the developmental process. Mutations in LAFL genes cause defects in desiccation tolerance, defects in cotyledon development of the zygotic embryo. On the other hand, ectopic expression of LEC-1 and LEC-2 causes somatic embryo generation on leaves and cotyledons of the Arabidopsis seedling. LEC-1 And LEC-2 plays role in regulating the expression of auxin responses and seed maturation and thus play a role in generating a totipotency state.LEC2 plays a pivotal role in activating the expression of the AGL15(Agamous-like)gene when overexpressed and thus plays role in enhancing S.E from immature zygotic embryos Overexpression of FUS3 and ABI3 does not cause somatic embryogenesis but however do give embryonic characteristic to seedlings.  AIL (Aintegeumenta-Like) genes take care of maintaining meristem.  Floral meristem, dividing tissues, root and shoot are the regions where AIL genes are expressed and these genes play role in maintaining meristematic state.

BABY BOOM(BBM) belongs to the clade of AP2/ERF TFs which also consist of AIL/PLT genes that act in a redundant manner to maintain the growth of the somatic embryos. Overexpression of BBM genes can cause regeneration in various other plant species.

 

Effect of Plant Growth Regulators:

A correct concentration ratio of cytokinin and auxin ratio for successful somatic embryogenesis. In many plant species cytokinin, auxin, abscisic acid are the key hormones for

generating embryogenic response and are responsible for regulating cell division and explant like zygotic embryo when placed in a medium containing 2,4-D leads to the formation of the somatic embryo. Explants when exposed to auxin for a longer period leads to somatic embryo formation and when exposed to a shorter period leads to adventitious shoot formation. Auxin response factors (ARF) are present in auxin-mediated plant development and this ARF binds to ARE(auxin response elements to mediate the expression of its target gene. About 22 ARF genes have been identified in Arabidopsis thaliana. Abscisic acid depending on the type of plant species can either have to promote or the inhibitory effect.

various  growth factors and genes in generation of somatic embryogenesis

various  growth factors and genes in generation of somatic embryogenesis

Image: (A) Showing impact on various  growth factors and genes in generation of somatic embryogenesis either directly or indirectly.

 

APPLICATIONS:

  • Somatic embryogenesis in studying changes the antioxidant activity of blueberry plant:

The Blue berry plant consists of high anti-oxidant property due to the presence of phenolic compounds which is consumed worldwide to because of its health benefits. Antioxidants scavenge the free radical and helps to prevent diseases like cancer, neurodegenerative diseases Studies on four different cultivars of blueberry showed that method of propagation has different effects on phenolic content. Leaf extracts from St.cloud and Northblue showed that donor plants had higher phenolic content than S.E generated plants Whereas it was seen that total phenolic concentration in S.E regenerated Chippewa was higher than the greenhouse grown donor plant.

 Thidiazuron(TDZ) induced somatic embryogenesis

Thidiazuron(TDZ) induced somatic embryogenesis

Conventional methods of cultivating blueberry is labour-intensive process and time-consuming process therefore invitro cultivation can be useful. Plant growth regulator TDZ is an effective

plant growth regulator. Invitro It is effective in inducing shoot proliferation and regeneration. It is also effective in the formation of somatic embryos when added to the growth medium.

Image: St. Cloud grown on a medium supplemented with TDZ. (a).Formation of protuberances (b)globular embryo(c) induction of heart shape embryo(d)torpedo-shaped somatic embryo

(e)epicotyl development. (f) germination of somatic embryos. (Ghosh.et.al)

 

2) Somatic Embryogenesis in the cultivation of date palm

S.E can be used for producing plant materials. P.dactylifera  explants like leaf bases, immature leaves, meristems can be used to generate callus by using a suitable medium containing sucrose. This callus is then transferred onto a medium supplemented with NAA to induce somatic embryo formation. The somatic embryos produced shoot after which shoots transferred into a media supplemented containing high sucrose concentration which promotes root formation. In-vitro plants whose generated roots are transferred to soil or some nursery  In-vitro propagation is useful in the multiplication of date palms. In this way date, palm industries can be expanded in terms of production in countries like Nigeria where Date palm is the main crop but the industry is small.

 

 

 

References:
  • Horstman, A., Bemer, M., & Boutilier, K. (2017). A transcriptional view on somatic embryogenesis. Regeneration, 4(4), 201–216. https://doi.org/10.1002/reg2.91

 

  • Ibrahim, K. M. (2010). THE ROLE OF DATE PALM TREE IN IMPROVEMENT OF THE ENVIRONMENT. Acta Horticulturae, 882, 777–778.

 

 

  • Krikorian, A. D., & Kaarina Simola, L. (1999). Totipotency, somatic embryogenesis, and Harry Waris (1893-1973). Physiologia Plantarum, 105(2), 347–354.

 

  • Finer, J. J. (1995). Direct Somatic Embryogenesis. In O. L. Gamborg & G. C. Phillips (Eds.), Plant Cell, Tissue and Organ Culture (pp. 91–102). Springer Berlin Heidelberg.

 

 

  • Ghosh, A., Igamberdiev, A. U., & Debnath, S. C. (2018). Thidiazuron-induced somatic embryogenesis and changes of antioxidant properties in tissue cultures of half-high blueberry plants. Scientific Reports, 8(1), 16978. https://doi.org/10.1038/s41598-018-35233-6

 

 

Ayesha Tungekar

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