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Growth
Regulators
and
Other
Factors
for
Somatic
Embryogenesis -
In most species an auxin (generally 2,4-0 at 0.5-5 mg/l) is essential for somatic embryogenesis. The auxin causes dedifferentiation of a proportion of cells of the explant, which begin to divide. In carrot, these small, compact cells divide asymmetrically, and their daughter cells stick together to produce cell masses called pro embryogenic masses or embryogenic clumps (ECs).
In the presence of auxin, the ECs grow and break up into smaller cell masses, which again produce ECs. But when the auxin is either removed or reduced (0.01-0.1 mg/l) and cell density is lowered, each EC gives rise to few to several SEs; each SE is believed to develop from a single superficial cell.
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The ability to regenerate SEs, i.e., totipotency, is acquired by cells during dedifferentiation in response to high auxin treatment but the mechanism is not well known. Some glycoproteins produced by totipotent cell masses are secreted into the medium; when these proteins are added into the culture medium they speed up the process of acquisition of totipotency. A class of proteins, called arabinogalactan proteins, induces SE regeneration in undifferentiated carrot cells, indicating their role in the process.
Auxins promote hypermethylation of DNA which may have a role in totipotency acquisition.
In many species like carrot, coffee; alfalfa etc., somatic embryogenesis is a two step process:
(i) SE induction on high auxin (up to 40-60 mg/l 2, 4 D) and
(ii) SE development on a low auxin or OR-free medium.
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In the SE induction phase, explant cells dedifferentiate, became totipotent and, in many species, form embryogenic clumps (ECs). Cells can be maintained in embryogenic stage on the induction medium for prolonged periods (over 10 years in carrot). When ECs are transferred from induction medium to an appropriate medium, SE differentiation proceeds from globular, heart-shaped, torpedo to cotyledonary stages; this is called SE development phase. Clearly in species like carrot, etc., OR requirements for the two phases are drastically different.
In most cases, SEs begin to germinate immediately after the cotyeledonary stage; this is called SE conversion. But often the plantlets so obtained are rather weak. It is, therefore, desirable to subject SEs to a maturation phase, following their development; in this phase the SEs usually do not grow but undergo biochemical changes to become more sturdy and hardy.
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SE maturation is achieved by culturing than on a high sucrose (up to 6% or even 40%) medium or in presence of a suitable concentration (0.2-0.4 mg/l) of ABA, or by subjecting them to partial desiccation. In most species, SE maturation improves their conversion, often by several-fold. In some species, e.g., Cicer arietinum, wheat etc., SE induction and development may take place on the same high auxin medium, although the frequency of mature embryos is rather low. In some species, SEs are produced in response to a cytokinin, e.g., BAP induces SEs in hypocotyls of young zygotic embryos of Trifolium sp., pea, etc.
But SEs are produced on immature cotyledons of these explants when 2,4-D is used in the .medium. It seems that cytokinins are effective in SE regeneration from embryogenic cells of young zygotic embryos, while auxins are effective on differentiated cells of both embryos and somatic tissues. Many workers have used combinations of auxins and cytokinins for SE regeneration in different species, but the role of cytokinin in these systems is not known.
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Other FactorsCertain other factors are reported to affect SE regeneration. For example, high K+ levels and low dissolved O2 levels promote SE regeneration in some species. In some other species, e.g. Citrus medica, some volatile compounds like ethanol inhibit SE regeneration. In soybean, low sucrose concentrations (5 and 10 g/l) promote SE regeneration as compared to high concentrations (20 and 30 g/l). In alfalfa, use of maltose as carbon source improves both SE induction and maturation (including germination) as compared to those on sucrose.
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