I was reading about using RNA-interference as a method of introducing pest-resistance in plants. In tobacco plants, using Agrobacterium vectors, genes specific to the nematode Meloidegyne incognitia were introduced. This was done in such a way that it produced both sense and anti-sense RNA in the host cell, forming dsRNA and ultimately silencing specific mRNA of the nematodes.
My question is, how can every single cell, or a proportion of cells sufficiently large so as to confer pest-resistance, of an already mature plant be transformed? Are a large number of vectors introduced, and as the infection gradually progresses, the immunity spreads throughout? Sorry if it's a stupid question, I've been out of touch with biology for a while.
Your inference is basically correct. The Agrobacterium acts as a vector for a plasmid containing the genetic construct one wishes to introduce into the plant genome. A medium containing a large number of bacterial cells transformed with this plasmid is injected into plant tissue, and the plasmids enter plant cells where parts of their DNA are integrated into the host genome. The overall effect of this is that there is localized integration in the areas where the Agrobacterium was introduced. A nice visual illustration of this comes from a paper from our lab where my colleagues introduced the genes for betalain biosynthesis into the tissue of Nicotiana benthamiana: in Figure 3 you will see only a localized area of cells around the injection site gain the ability to produce betalains.
You can find a nice review of the whole process of transformation with Agrobacterium here.
Model of development reveals shapes of cell lineages and links to regeneration
Figure 1 (A): An example generative model showing an organism with N = 3 genes and two cell types. Circles represent all possible cell types. The organism is composed of cell types represented by white circles and does not contain the gray cell types. Binary strings written inside the circles represent the presence (1) or absence (0) of determinants in those cell types. (B) Schematic of “organismal development” in the model. All cell types synchronously undergo cell division, exchange signals, and respond to signals through gene regulation until reaching a steady state. (C) Lineage graph of the homeostatic organism in A. Credit: Institute for Basic Science
Various forms of complex multicellular organisms have evolved on Earth, ranging from simple Volvox carterii, which possess only two cell types, to humans, with more than 200 cell types. All originate from a single zygote, and their developmental processes depend on switch-like gene regulation. These processes have been studied in great detail within a few model organisms such as the worm C. elegans, and the fruit fly D. melanogaster. It is also known that the key molecules and mechanisms that are involved in the development of multicellular organisms are highly conserved across species.
What is also remarkable is that only a handful of molecules and mechanisms that go into the development of a multicellular organism can generate such a huge diversity of forms and complexity. Recently, researchers from the Center for Soft and Living Matter within the Institute for Basic Science investigated how this is possible using a simple mathematical model. Through this work, they sought to answer two seemingly opposite questions: what are the limits of diversity that can be generated through development, and what common features are shared among all multicellular organisms during their development.
Three processes are common to biological development in all multicellular organisms: cell division, cellular signaling, and gene regulation. As such, this study's model generated millions of these rules and explored them in an unbiased way. The mappings generated by the model represent how one cell type converts into another during the lifetime of the organism. Traditionally, previous cell type maps based on single-cell transcriptomics are biased to be tree-like, with stem cells sitting at the root of the tree, and increasingly more specialized cells appearing downstream along the branches of the tree. However, the cell-type maps produced by the new mathematical model were far from tree-like it was found that there were many cross-links between different branches of the cell types. These resulted in directed acyclic graphs, and tree lineages were found to be the least prevalent. This means that it is possible for multiple developmental routes to converge on the terminal cell type in the maps generated by the model.Figure 2: (A) Lineage graph from real organisms. (B) Examples of unicellular, cyclic, chain, tree, and directed acyclic lineage graphs generated from the mathematical model. Credit: Institute for Basic Science
Surprisingly, it was also found that many organisms produced by the mathematical model were endowed with the ability to regenerate lost cells, without any selection imposed by the authors. When a single cell type is isolated from the adult organism, single cell could transform into and replenish all the other cell types. This ability to generate all the cells of the body is called pluripotency, and these cells granted the organisms in the model the ability of whole-body regeneration. Interestingly, most tree-type lineages contained few pluripotent cells, in comparison to other graph types.
While mammals, including humans, are especially bad at regenerating damaged parts, many animals such as worms and hydra, are exceptionally good at this ability. In fact, whole-body regeneration occurs widely across the multicellular animal tree of life, and therefore it has been hypothesized that whole-body regeneration could be an epiphenomenon of biological development itself. The fact that pluripotency occurred in this very simplified model suggests that this trait is indeed likely to emerge due to the process of development itself, and no special extra components are required to put it in place.
In addition to these results, it is anticipated that the framework of this model can be used to study many more aspects of development. This generative model is simple and modular, and it can be easily expanded to explore important processes which were not included in the present study, such as the effect of the spatial arrangement of cells and the effect of cell death. The researchers further described some possible real-life experiments to test some of the predictions made by their mathematical model. It is hoped that the framework of this model will prove useful for uncovering new features of development, which may have a wide range of implications in developmental biology and regenerative medicine.
Development of Primordial Soup Theory
In 1924, Alexander Ivanovich Oparin, proposed that abiogenesis did occur in a warm water body like a pond or an ocean on primordial Earth. He explained that the conditions present then, could have spontaneously given rise to simple and crude life forms. The lack of experimental proof could be explained by the fact that not only had the Earth’s environment changed radically since then, but also that any new life forms would immediately be consumed by present-day organisms inhabiting that area. He hypothesized that the current level of atmospheric oxygen prevents the synthesis of organic compounds essential to the generation of life, and that a “primeval soup” of organic bio-molecules could be generated by the action of sunlight in an atmosphere devoid of oxygen. These bio-molecules would then combine with themselves and grow till a critical mass was attained, after which, it would divide via fission into daughter molecules. This mechanism would form the basis for a primitive form of metabolism.
At around the same time, a British biologist, J. B. S. Haldane, introduced his concept of the chemical origin of life. He likened the primitive ocean to a vast chemical laboratory, one that possessed a mix of inorganic compounds. Under the action of sunlight, the atmosphere of carbon dioxide, ammonia, and water vapor would allow the transformation of inorganic molecules to organic molecules (primitive life forms). These molecules would then interact with each other to produce complex compounds, which would, in turn, form cellular components. Eventually, a membrane structure would be formed, and this would then evolve to encapsulate self-replicating molecules, thereby, producing the first living cell. This hypothesis for the origin of life was termed as biopoiesis by J.D. Bernal.
The development of a complex, multicellular organism from a single-celled fertilized egg is a miraculous transformation that has been the subject of intense study for over a hundred years. The Developmental Biology research group consists of a diverse group of interactive faculty investigating a wide variety of development processes. We use several well-studied experimental organisms including invertebrates such as the soil nematode Caenorhabditis elegans and the fruitfly Drosophila melanogaster, vertebrates such as zebrafish (Danio rerio) and mouse (Mus musculus), and plants Populus and Arabipdosis, as well as the bacteria Myxococcus xanthus and Pseudomonas aeruginosa.
Specific aspects of development that are being studied include the role of chromatin remodeling in maintaining cell fate decisions (Dorus, Hall, MacDonald, Maine, Raina), signal transduction mechanisms that control germline development (Belote, Dorus, Maine, Pepling), development and aging of the vertebrate nervous system (Gold, Korol, Lewis, MacDonald), plant cell wall development and reproductive timing (Coleman, Raina), and formation of bacterial biofilms (Garza, Welch).
The group uses a wide range of approaches—including imaging, genetics, molecular biology, and bioinformatics—to address important questions in the field. Members of the our group interact with researchers from neighboring campuses in Syracuse that have a shared interest in developmental biology through monthly meetings of the Developmental Biology Interest Group (DBIG).
The origins of multicellular organisms
Multicellularity has evolved in several eukaryotic lineages leading to plants, fungi, and animals. Theoretically, in each case, this involved (1) cell-to-cell adhesion with an alignment-of-fitness among cells, (2) cell-to-cell communication, cooperation, and specialization with an export-of-fitness to a multicellular organism, and (3) in some cases, a transition from “simple” to “complex” multicellularity. When mapped onto a matrix of morphologies based on developmental and physical rules for plants, these three phases help to identify a “unicellular ⇒ colonial ⇒ filamentous (unbranched ⇒ branched) ⇒ pseudoparenchymatous ⇒ parenchymatous” morphological transformation series that is consistent with trends observed within each of the three major plant clades. In contrast, a more direct “unicellular ⇒ colonial or siphonous ⇒ parenchymatous” series is observed in fungal and animal lineages. In these contexts, we discuss the roles played by the cooptation, expansion, and subsequent diversification of ancestral genomic toolkits and patterning modules during the evolution of multicellularity. We conclude that the extent to which multicellularity is achieved using the same toolkits and modules (and thus the extent to which multicellularity is homologous among different organisms) differs among clades and even among some closely related lineages.
DESIGN AND METHODS
The original version of Biofundamentals (MCDB 1111 Figure 1) was approved by the department and College of Arts and Sciences at the University of Colorado–Boulder (UC Boulder) as a four-credit course that replaced the existing versions of two courses: Introduction to Cell and Molecular Biology (MCDB 1150) and the accompanying laboratory (MCDB 1151). Subsequently, the course was taught as MCDB 1150–section 3, a three-credit course that met three times a week for 50 min. While we have not archived the course’s Web-page history, we have preserved the last Web version, which reflects the course’s original organization. Approximately one Web page was assigned for each course period. 2 In the last four years (2011–2015) the Web-based materials were first adopted to be read by students (assigned randomly to groups of five to eight students each) using the interactive system developed by Highlighter.com. With the demise of Highlighter.com in 2013, students were assigned readings from the Biofundamentals text using the nota bene system (Zyto et al., 2012). 3 As before, students were assigned at random into groups of five to eight students each. Beginning in 2012, students were also asked to complete a beSocratic activity before each class (the system was running on servers at Michigan State University). Both activities were mandatory for students in the class, with compliance influencing students’ final grades. The use of deidentified beSocratic data and nota bene data were judged exempt by the UC Boulder Institutional Review Board (IRB 0304.09 and 15–0347). Student responses to beSocratic activities were characterized based on predefined rubrics to identify apparent issues in understanding underlying ideas. In addition, questions raised during in-class discussions were noted and used to inform subsequent revisions of first the website and then the text and associated beSocratic activities. Similarly, exam questions were analyzed using a rubric (three-dimensional learning assessment protocol [3D]-LAP) generated in the course of American Association of Universities (AAU)-funded studies at Michigan State University to characterize the nature of exam questions on the basis of a number of criteria, including whether the answers involved disciplinary core ideas and cross-cutting concepts and practices (Laverty et al., 2016) these rubrics are illustrated for the final exam given in 2015 (see Supplemental Material 3). The course was routinely taught with the aid of learning assistants (Otero, 2006), undergraduate students who had previously taken the course, who were taking a concurrent course in science pedagogy, and who met weekly with the course instructor to discuss students’ issues.
FIGURE 1. Schematic of the design progression from a conventional introductory molecular biology course to Biofundamentals, which has a largely flipped and interactive course design focused on a set of core concepts. At various steps in the process, observations from student interactions with course materials led to revisions in the representation of materials.
Freeloaders Are Everywhere
What is more, within a biofilm, some bacteria grow faster if they do not respond to chemical signals produced by their neighbors.18 This apparently allows them to grow with less restraint than the rest of the biofilm population. Also, some bacterial populations release polysaccharides so they can form mats on liquid surfaces (a type of biofilm), which increases the population’s access to oxygen. Within this mat though, a few individual cells will stop making the polysaccharide.19 This allows them to benefit from the mat without expending the energy to help make the mat.
Such "cheaters" take advantage of the cellular activity of others without expending energy to contribute to the community. In fact, "cheating" is recognized as a major problem in the evolution of social cooperation.20 Why do the work when others are doing the work for you?
Another major problem is that selection at the cell-level and selection at the multicellular organism-level are not equivalent. In fact, they are virtually opposite. If selection is operating at the multicellular level, it cannot simultaneously be operating at the individual cell level.21 Single cells thrive by reproducing more than their neighbors, while cells in a multicellular organism coordinate their reproduction. Thus, Darwinian views of natural selection are inconsistent with the evolution of multicellular organisms. The “selection” that supposedly formed and maintained the unicellular world for billions of years would be in diametric opposition to any “selection” supposedly attempting to form a new multicellular world.
This brings us back to my initial question—why even evolve multicellular systems?
The formation of multicellular organisms means that cells must relinquish their unicellular, programmed behavior in favor of a coordinated behavior. Why they would do this is currently unanswerable. How they would do this is also currently unanswerable, but certainly would be an enormously complicated transformation one that is clearly far from “easy.”
What is more, any multicellular evolution almost certainly would require the formation of new genes. The development of multicellular biology requires that all the cells of the organism “have the same set of genes and obey the same rules.”22 Not only do new genes need to form during multicellular evolution , but the same genes and regulatory controls need to form in all the cells of the multicellular system. This is necessary to provide the new proteins and genetic activity required by multicellular organisms. Without new genes, single cells would remain single cells.
Viewing Mites under Stereo Microscope
- House dust (dust can also be collected from head pillows)
- Stereo microscope
- Saturates sodium chloride solution
- 45um mesh sieve
- Crystal violet
- Place 0.05 grams of the dust sample in 30 mL of sodium chloride solution a
- Add five drops of detergent
- Subject the dust to 20 minutes of ultrasonic treatment
- Rinse the suspension using 45um mesh sieve
- Stain using crystal violet
- View under stereomicroscope
* Alternatively, dust mites can be viewed by simply placing a small amount of the dust on water surface and observing under 20x magnification.
As for the larger multicellular organisms, they can be viewed under the microscope by simply placing the organism (insect, leaf etc) under the stereomicroscope and adjusting the magnification to view the surface of the organisms.
Transformation of a multicellular organism - Biology
Multicellular organisms contain a vast array of highly specialized cells. Plants contain root cells, leaf cells, and stem cells. Humans have skin cells, nerve cells, and sex cells. Each kind of cell is structured to perform a highly specialized function. Often, examining a cell's structure reveals much about its function in the organism. For instance, as we have already seen, epithelial cells in the small intestine are specialized for absorption due to the numerous microvilli that crowd their surfaces. Nerve cells, or neurons, are another kind of specialized cell whose form reflects function. Nerve cells consist of a cell body and long processes, called axons, that conduct nerve impulses. Dendrites are shorter processes that receive nerve impulses.
Sensory cells&mdashthe cells that detect sensory information from the outside environment and transmit this information to the brain&mdashoften have unusual shapes and structures that contribute to their function. The rod cells in the retina of the eye, for instance, look like no other cell in the human body. Shaped like a rod, these cells have a light-sensitive region that contains numerous membranous disks. Within each disk is embedded a special light-sensitive pigment that captures light. When the pigment receives light from the outside environment, nerve cells in the eye are triggered to send a nerve impulse in the brain. In this way, humans are able to detect light.
Source: Lerner, K L and Lerner, B W (eds) The Gale Encyclopedia of Science. Vol. 1. 3rd ed. Detroit: Gale,2004. p781 [accessed: June 9, 2017 via National Library of Australia]
Notes on Cellular Differentiation | Cell Biology
Differentiation is the process by which the genes are preferentially active and the gene products are utilised to bring some phenotypic changes in the cell. It is not the only property of multicellular organisms. Many unicellular organisms undergo phenotypic changes along with changes in physiological processes.
Any change in the environment of unicellular or­ganisms whether—physical or at the nutrient level—can undergo remarkable physical cellular changes like the formation of different types of spores, sporulation in bacteria, fungi etc.
These are the example of cellular differentiation in unicellular organisms. The differentiation observed in higher or­ganisms, particularly animals, is different and complicated. It has attracted developmental bi­ologists to study the development of an embryo from a single cell, i.e., zygote.
This process takes place in several steps:
Development of zygote to form blastula (group of undifferentiated cells).
Differentiation and move­ment of cells to form specialised cell layers.
Development of specialised cells into tissues, organs and growth of the embryo.
v. Growth, Maintenance and Regeneration of some cells.
Molecular Changes During Oogenesis and Fertilisation:
The process of female gamete formation is known as oogenesis. The primordial germ cells are called oogonia. When the oogonia start meiosis they are called oocytes where an exten­sive growth phase occurs.
During this growth phase of oocyte, large number of metabolic and morphological changes occur.
i. Increase of RNA synthesis—along with polytene-like changes, are found in the chromosome.
ii. Amplification of Ribosomal genes takes place leading to the increased synthesis of ribosomal RNA. Associated cytological changes are the occurrence of many nucle­oli in the nucleus.
iii. Size of the nucleus is increased indicating the metabolic changes of the nucleus.
iv. Accumulation of protein, lipid and carbo­hydrate takes place which will be utilised during the formation of embryo. In higher animals all those nutrients are produced by the liver and follicle cells of the developing oocyte. However, mammalian oocytes do not accumulate yolk proteins as they come through mother’s bloodstream and thus storage of nutrients is not necessary here.
v. In non-mammalian oocyte the asymmetry in polarity is found during its development. One end of the cell is called the Vegetal pole containing most of the nutrients and yolk platelets. The other end is called the Animal pole containing ribosomes and mitochondria besides nucleus.
The embryo is developed at this end. After these developmental changes, the meiosis takes place in the oocyte to produce mature egg which is a highly differentiated specialised cell.
increase their number by mitosis and then undergoes meiosis to form sperms. Cellular and other molecular changes are not so significant like oogenesis. Considerable changes are found after fertilisation during the development of embryo.
Role of Cytoplasm in Cellular Differentiation:
The importance of cytoplasm on cell differen­tiation has been demonstrated in large number of experiments.
The egg of snail produces a lobe-like struc­ture at the vegetal end during cell differentia­tion which is known as Polar lobe. If this lobe is excised, defective embryo is produced. Again, a coloured area is produced in the amphibian egg during cell differentiation after fertilisation. This is known as Grey Crescent. If the grey crescent is injured it induces abnormality in the nervous system.
In case of amphibian egg, if the first cleavage is perpendicular to the grey crescent and the resulting blastomere is separated, each blastomere will produce a normal animal. But if the cleavage takes place parallel to the grey crescent and if the two blastomeres are separated—the one having grey crescent will produce normal animal. Thus the differentiation of cell depends on the partitioning of substances in the cyto­plasm.
Another observation has been made in case of the embryonic development of egg of the round worm, Ascaris. During the development of embryo, the first cleavage occur perpendicular to the animal vegetal axis.
When the animal pole of the new blastomere starts dividing, the heterochromatic portion of the chromosome becomes degenerated. The euchromatic portion of the chromosome is fragmented into numerous small chromosomes by a process known as chromosome diminution.
Thus chromosome diminution occurs during embryonic develop­ment. Now Theodor Boveri made an exper­iment in which eggs are centrifuged before cleavage in order to disturb the polarity of the cell.
By centrifugation, the mixture of both animal and vegetal cytoplasm occurs and the first cleavage occurs along the animal vegetal axis not perpendicular to it. No chromosome diminution occurs—this indicates the role of cytoplasm in chromosomal behaviour.
Again, in case of embryonic development of Drosophila, the primordial germ cells generally arise from the posterior end of the egg. Illmensee and Mahowald made an experiment by removing some cytoplasm from the posterior end of the egg of Drosophila and injected then into the anterior end of another egg. It has been noted that germ ceils are then produced from the anterior end of the injected egg.
Thus it can be said that the cytoplasm has an important role in inducing differentiation in cells. Its role has also been noted in adult cells. When the inactive nuclei of mature erythrocytes are injected into the cytoplasm of active cells, the inactive nuclei become transcriptionally active.
Similar effect has also been noted in other types of cells. All these results clearly reveal that cytoplasmic factors are responsible for cell differentiation. But in case of mammals, embryonic cells are totipotent after the first cleavage division till the development of the embryo.
This has been evidenced by segregating mouse or rabbit embryo at 8 or 16 cells stage. Each blastomere gives rise to blastocyst if the proper cultural conditions are provided and these blastocysts will form normal animal after implantation into the uterus of another female rat. This property of totipotency of egg cell is due to the homogeneity of its cytoplasm.
The retention of totipotency by an individual cell has been demonstrated in case of plant systems. Any cell from any part of the plant can be grown in culture where they form at first the mass of undifferentiated tissue, known as callus.
This callus can be regenerated into a whole plant if the proper concentration of the hormone is supplemented in the media. But single animal cells after the first cleavage division will not form full animal even if the proper nutrients and other conditions are given in culture.
Genetic totipotency of animal cell has been demonstrated by John Gurdon in the nuclear transplantation experiment in toad showing that each and every cell possesses all the genes required for the development of the whole or­ganism.
Still the development or differentiation into whole organism has not been in animal sys­tem. Thus researchers thought that the pattern of gene expression in each cell type is different, although all the genes are present in each cell. So we see that the control of gene expression in higher organisms, particularly animals, is very complicated.
In other words, many types of post-transcriptional modifications of the pri­mary transcript of the gene are regulated in the formation of the final functional gene product necessary for a particular cell differentiation or development.
Two model organisms are used for the study of the specific genes involved for a particu­lar cellular development. One is the round­worm, Caenorhabditis elegans, and another is Drosophila, C. elegans.
These are used as a model in the developmental genetics for the following reasons:
i. Short life cycle (3 days).
ii. Ease of maintenance like E. coli.
iii. Can reproduce by self or by cross- fertilization.
iv. Hermaphrodite (XX)—contains 5 pairs of autosomes and 1 pair of X chromosomes.
v. The male (XO) contains 5 pairs of auto­somes and a single X chromosome.
vi. Haploid genome is about 8 x 10 7 bp.
vii. More than 600 genes have been identified.
viii. Easy to obtain homozygous populations, as self-fertilization is possible.
ix. In-breeding is automatic in hermaphrodite population.
x. DNA transformation through microinjec­tion at the selected stage of development is possible in this animal.
The reproductive system of the hermaphro­dite animal produces a bilobed structure in early stages of development. The oocytes and embryos in each lobe start developing from the distal end to the uterus. All stages can be detected easily and thus microinjection of DNA can be done at the selected stage of development.
Another important work on Drosophila made the discovery of new class of genes called Homeotic genes. These genes were identified through mutations where one part of the body is replaced by a structure that is found somewhere else. This unusual thing occurs during development of the embryo. The mutations of homeotic gene (Antp) result in the formation of middle legs in place of antenna of Drosophila.
The molecular analysis of homeotic gene has resulted in the discovery of a 180bp sequence present in other homeotic genes. This sequence is known as Homeobox. Since the homeobox is present in higher animals also, gene cloning and sequencing has been done from mice to man. The function of these genes has been found to be the same as in Drosophila.
The homeobox genes are expressed in highly differentiated cells in case of Amphibians and Mammals. Home­obox genes are found to play very important role in developmental processes and they are highly conserved during evolution.
Homeotic Genes of Cellular Differentiation:
During embryonic development in Drosophila, the identification of different segments of the body has been found to be under the control of many genes. Two complexes of these genes have been identified in Drosophila.
One is Antennapedia Complex (ANT-C) located in the chromosomal position 84AB. Another group of genes is known as Bi-thorax complex (BX-C) located at chromosomal position 89E. The first group (ANT-C) is responsible for the develop­ment of the head and thoracic segments while the BX-C are responsible for the development of the trunk segments.
The analysis of this homeotic complex has been done in Beetle also. One of the important features of these genes is the large size—about 50 to 100 kb and very large introns. The next important feature is the presence of con­served region, i.e., Homeobox.
The homeobox generally codes for a DNA binding protein domain, whose product binds to DNA. Another important characteristic is the presence of cis- acting regulatory regions.
Cellular differentiation, pattern formation and morphogenesis were studied in detail in case of plant Arabidiopsis as model system. It involves some factors which help in causing different cell types, organs etc. to originate at specific locations. This is also done by cell shape changes and planes of cell division. Cell differentiation is clearly noted during embryo- genesis.
Different and molecular observations were studied in maize and rice in case of monocotyledonous plants. In dicots, several genes involved in storage proteins have been cloned and their expressions noted in Soybean. However, detailed studies have been done in Crucifers, particularly in Arabidiopsis thaliana, in identifying embryo developmental genes but mutagenesis.
Arabidopsis has been accepted as a model system in having the following criteria:
i. Small size of the plant.
ii. Small size of the nuclear genome.
iv. Self-fertilizing bisexual flowers.
v. Large number of progeny in each flower.
vi. Starchy endosperm is absent.
Molecular studies show that a number of genes are involved during early and late stage embryogenesis in both zygote and somatic em­bryo. Different cell division patterns have been noted in somatic embryos. Many pattern genes have been identified in Arabidopsis through mutagenesis, which show seedling abnormali­ties.
These mutants were named gnom (gn), affecting the apical and basal region, gurke (gk) affecting the apical region, faeckel (fx) affecting the central part of the seedling. There is the other mutants which change the shape of the seedling.
Several methods have been used for cloning pattern genes which has been used as an in­serted probe to isolate the flanking DNA in Arabidopsis. T-DNA tagging approach is used to clone the first embryo pattern gene of Ara­bidopsis. The study of plant developmental genetics and cell differentiation have been changed con­siderably with the isolation of several mutant phenotypes and biochemical mutants in Ara­bidiopsis.
More than 500 embryo-defective mutants and many mutants showing aberration in vegetative development have been analysed in Arabidiop­sis. Arabidiopsis Biological Resource Centre has been established in the Ohio State Univer­sity for the supply of mutant seeds and DNA.
Some of these mutants have been found to alter in basic cellular functions necessary for normal growth and development, i.e., the function of ‘house-keeping genes’.
The molecular basis of these mutants have also been studied to find out the relationship between gene function and nor­mal development. The first known biochemical mutant noted in Arabidiopsis is bio I auxotroph (mutant 122 G-E).
This mutant bio I stops growth of the embryo at the heart stage of development but shows normal growth when biotin is added in the medium. The normal bio-synthetic pathway of biotin in bacteria is shown in Fig. 19.1.
It has been noted that bio I mutants were defective in the synthesis of 7, 8- diaminopelargonic acid from 7-keto 8 diaminopelargonic acid. This step is regulated in bacteria by bio A gene. When the bio A gene from bacteria is introduced into the bio I mutant plants, the transgenic plants do not require biotin for their growth.
Thus this experiment has shown that a plant mutant can be recovered to normal by the introduction of cloned bacterial gene.
Another mutant ‘gnom’ or emb 30 shows another type of defect in basic cellular function. Meinke (1985) first observed the morphologi­cal structure as fused cotyledons and rootless plants which can be grown in culture. He also identified the first allele (112A-2A) of this mutant.
Many other alleles have been identified in other laboratories. This mutant and other embryo-defective mutants showed alterations in embryonic pattern formation and altered pattern of cell division during embryo development.
DNA sequence analysis of this mutant (EMB 30) shows homology with the SEC 7 gene of yeast which helps in the protein transport from the Golgi, indicating its role in transporting some proteins to the cell surface. It may also have an alteration in the signal transduction pathway.
Another interesting mutant is ‘fusca’ which shows insufficient accumulation of anthocyanin in developing cotyledons. Seeds of this mutants germinate to produce defective seedling which fail to complete the life cycle. Sev­eral genes of ‘fusca’ mutants have now been cloned and sequenced. These are: FUSI/COPI FUS2/DETI, FUS6/COP11, FUS7/COP9 etc.’ These genes encode some novel proteins.
The product of COPI gene has N-terminal zinc binding domain and a C-terminal domain which shows homology with the B-sub-unit of G” proteins. Again, the sequence of FUS6 gene shows similarity with that of human gene GPSI. Thus, it can be said that G proteins also play an important role in signal transduction pathways of plants.
The pattern formation and morphogenesis go hand in hand during development in multicel­lular organisms. Cyto differentiation is nothing but a division of labour between component cells. During cyto differentiation, some alter­ations in the biochemical and structural prop­erties occur leading to functional specialisation.
In the developmental stage the formation of cell diversity is the process of cell pattern or shape formation where positional information determines the final development of a cell. In the plant systems, the presence of any devel­opmental memory has not been clearly pointed out.
But the involvement of some localised activ­ity of specific regulatory proteins in Cyto differentiation and development has been estab­lished. It has been observed that the de­velopment of floral organs in Arabidiopsis is regulated by some genes encoding transcription factors.
The generation of individual cell types within an organ is dependent on the cell autonomous expression of regulatory molecules. Larkin (1993) has shown the role of transcription factors in trichome differentiation.
The mutational studies have shown that mutations in a specific gene can inhibit the development of individual cellular domains, keeping the other domains normal. For example, monopteros mu­tation shows no development of hypocotyl and root but the shoot meristems and cotyledons are not affected.
But there are some mutants like emb 30/gnom, hydra/fuss rootless and ‘monopteros’ which show defective shape and also abnormalities in cell differentiation, par­ticularly in vascular tissue organisation.
That means the respective gene products are es­sential both for morphogenesis and cellular differentiation. Thus, the relative positioning of cells in plant development is very important to continue the cell-cell signalling events during morphogenesis.