Stages Of Microspore Embryogenesis Research Articles

Phospholipase C is a novel regulator at the early stages of microspore embryogenesis in Nicotiana tabacum

ABSTRACT Microspore transfers the developmental fate into embryogenesis in vitro regulated by determinant factors of stress-induced. However, the key regulators of microspore embryogenesis (ME) are still largely undiscovered to reveal the mechanism of cell fate transition. Here, we report that Phospholipase C (PLC) is involved at the early stages of ME in Nicotiana tabacum. NtPLC2/3/4 are expressed at the initial stages of ME. The expression levels of NtPLC2/3 are transient activated after 3 days in culture, while the expression level of NtPLC4 maintains relatively stable. Inhibition of PLCs induces the decrease in NtPLC2/3/4 expression level and decline of ME yield. We confirm that lipids in microspore are degraded and then re-accumulate at first embryonic division stage. Inhibition of PLCs suppresses the lipids metabolism at the early stages of ME. Thus, we propose that PLCs-mediated lipid metabolism is a novel regulator at the early stages of ME.

Gametic embryogenesis and callogenesis in Isolated microspore culture of Jatropha curcas L. a recalcitrant bioenergy crop

Jatropha curcas is an undomesticated crop and its plantations did not meet commercial expectation due to absence of high yielding commercial line with desired agronomic characters. Earlier, breeding efforts did not pay much attention on yield and disease improving traits due to lack of desired genetic variability. Isolated microspore culture is one of the most widely used in vitro techniques to induce gametic embryogenesis with wide degree of genetic variability for the development of haploids and doubled haploids. In this study, an efficient isolated microspore culture system was established for different genotypes with the optimization of various factors that affect microspore embryogenesis. After 15–20 days of culture at 25 °C, tetrads, mid, early un-vacuolated and vacuolated late stage uninucleate microspores, which were preincubated at 4 °C for 7 days under shaking conditions, induced the formation of embryo like structures (ELSs) in a modified MS medium with 2.0 mg/l, 2,4-d, 0.1 mg/l kinetin, 300 mg/l casein hydrolysate, 1 g/l glutamine, 0.5 mg/l folic acid, 0.05 mg/l biotin and 5% sucrose. It is observed that the shock at 4 °C for 7 days, subsequently incubation of microspore cultures at 25 °C for 15 days and then at 15 °C for 10 days played a significant role in induction and accelerated creation of ELSs in three different genotypes. Microscopic analyses confirmed the different developmental stages of microspore embryogenesis including cell division and multicellular embryo like structures. Additionally, flow cytometric analyses verified the calli and ELSs were haploids in nature and strengthened that the origin was from microspores. This study supports the evidence of gametic embryogenesis in Jatropha microspore culture. Development of haploid and doubled haploids in Jatropha Microspore culture.

Dynamic Changes in Arabinogalactan-Protein, Pectin, Xyloglucan and Xylan Composition of the Cell Wall During Microspore Embryogenesis in Brassica napus.

Microspore embryogenesis is a manifestation of plant cell totipotency whereby new cell walls are formed as a consequence of the embryogenic switch. In particular, the callose-rich subintinal layer created immediately upon induction of embryogenesis was recently related to protection against stress. However, little is currently known about the functional significance of other compositional changes undergone by the walls of embryogenic microspores. We characterized these changes in Brassica napus at different stages during induction of embryogenic microspores and development of microspore-derived embryos (MDEs) by using a series of monoclonal antibodies specific for cell wall components, including arabinogalactan-proteins (AGPs), pectins, xyloglucan and xylan. We used JIM13, JIM8, JIM14 and JIM16 for AGPs, CCRC-M13, LM5, LM6, JIM7, JIM5 and LM7 for pectins, CCRC-M1 and LM15 for xyloglucan, and LM11 for xylan. By transmission electron microscopy and quantification of immunogold labeling on high-pressure frozen, freeze-substituted samples, we profiled the changes in cell wall ultrastructure and composition at the different stages of microspore embryogenesis. As a reference to compare with, we also studied in vivo microspores and maturing pollen grains. We showed that the cell wall of embryogenic microspores is a highly dynamic structure whose architecture, arrangement and composition changes dramatically as microspores undergo embryogenesis and then transform into MDEs. Upon induction, the composition of the preexisting microspore intine walls is remodeled, and unusual walls with a unique structure and composition are formed. Changes in AGP composition were related to developmental fate. In particular, AGPs containing the JIM13 epitope were massively excreted into the cell apoplast, and appeared associated to cell totipotency. According to the ultrastructure and the pectin and xyloglucan composition of these walls, we deduced that commitment to embryogenesis induces the formation of fragile, plastic and deformable cell walls, which allow for cell expansion and microspore growth. We also showed that these special walls are transient, since cell wall composition in microspore-derived embryos was completely different. Thus, once adopted the embryogenic developmental pathway and far from the effects of heat shock exposure, cell wall biosynthesis would approach the structure, composition and properties of conventional cell walls.

Novel features of Brassica napus embryogenic microspores revealed by high pressure freezing and freeze substitution: evidence for massive autophagy and excretion-based cytoplasmic cleaning

Induction of embryogenesis from isolated microspore cultures is a complex experimental system where microspores undergo dramatic changes in developmental fate. After ~40 years of application of electron microscopy to the study of the ultrastructural changes undergone by the induced microspore, there is still room for new discoveries. In this work, high pressure freezing and freeze substitution (HPF/FS), the best procedures known to date for ultrastructural preservation, were used to process Brassica napus microspore cultures covering all the stages of microspore embryogenesis. Analysis of these cultures by electron microscopy revealed massive processes of autophagy exclusively in embryogenic microspores, but not in other microspore-derived structures also present in cultures. However, a significant part of the autophagosomal cargo was not recycled. Instead, it was transported out of the cell, producing numerous deposits of extracytoplasmic fibrillar and membranous material. It was shown that commitment of microspores to embryogenesis is associated with both massive autophagy and excretion of the removed material. It is hypothesized that autophagy would be related to the need for a profound cytoplasmic cleaning, and excretion would be a mechanism to avoid excessive growth of the vacuolar system. Together, the results also demonstrate that the application of HPF/FS to the study of the androgenic switch is the best option currently available to identify the complex and dramatic ultrastructural changes undergone by the induced microspore. In addition, they provide significant insights to understand the cellular basis of induction of microspore embryogenesis, and open a new door for the investigation of this intriguing developmental pathway.

Hordeins are expressed in microspore-derived embryos and also during male gametophytic and very early stages of seed development

Microspore-derived embryos induced by anther or isolated-microspore culture display certain characteristics of zygotic embryos. Furthermore, the expression of certain endosperm genes has been described in these non-zygotic embryos. The expression of hordein genes encoding the main barley endosperm proteins has been studied using a wide range of methods (RT-PCR, in situ hybridization, ELISA sandwich, western blotting immunocytochemistry, and cytochemistry) to ascertain their presence or absence during the induction and first stages of microspore embryogenesis. Due to the very sensitive techniques used it was possible to detect for the first time hordein expression during microspore embryogenesis. Surprisingly, these hordeins were also detected at different stages of male gametophytic development as well as during the very early stages of seed development, when they have not hitherto been detected. The expression and localization of these storage proteins and their corresponding transcripts provide new information about barley microspore embryogenesis and its relationship to zygotic embryogenesis. Although only small quantities of hordeins are accumulated during microspore embryogenesis they seem to be necessary for the initial development of the microspore-derived embryo. This idea is supported by the changes detected in their concentration throughout this process and is in accordance with previously published data concerning the importance of endosperm proteins for embryo development in both microspore culture and in planta.

Genes normally expressed in the endosperm are expressed at early stages of microspore embryogenesis in maize.

Reproduction in flowering plants is characterized by double fertilization and the resulting formation of both the zygotic embryo and the associated endosperm. In many species it is possible to experimentally deviate pollen development towards an embryogenic pathway. This developmental switch, referred to as microspore embryogenesis or androgenesis, leads to the formation of embryos similar to zygotic embryos. In a screen for genes specifically expressed during early androgenesis, two maize genes were isolated by mRNA differential display. Both genes represent new molecular markers expressed at a very young stage of androgenic embryogenesis. When their expression pattern was studied during normal reproductive development, both showed early endosperm-specific expression. Investigation of the cytological features of young androgenic embryos revealed that they present a partially coenocytic organization similar to that of early endosperm. These findings suggest that maize androgenesis may possibly involve both embryogenesis and the establishment of endosperm-like components.

Characterization of cDNAs expressed in the early stages of microspore embryogenesis in barley (Hordeum vulgare) L.

To gain insight into the molecular events occurring in the very early stages of barley microspore embryogenesis, cDNA clones corresponding to genes differentially expressed during the early stages of microspore culture were isolated and characterized. A cDNA library established from barley microspores cultured for three days was differentially screened against probes generated from freshly isolated microspores. Three cDNAs representing genes not previously identified in barley were isolated. ECA1 (early culture abundant 1) lacked significant homology to known genes or proteins, and the transcript was only expressed during the early stages of culture. Expression was also reduced in low-density control cultures, therefore this gene may play a role in the early stages of barley microspore embryogenesis. ECGST (early culture glutathione S-transferase) had homology to parA-like genes, which are members of a newly discovered group of glutathione S-transferases (GSTs). The protein corresponding to ECGST may be important in protecting cells from oxidative stress during the culture process. ECLTP (early culture lipid transfer protein) had homology to lipid transfer proteins (LTPs), and had an expression pattern similar to that of an LTP known to be a marker of the early stages of embryogenesis in the carrot somatic embryogenesis system. The identification and characterization of the clones isolated in this study provides new information on the events involved in barley microspore embryogenesis.