Journal of Experimental Botany, Vol. 51, No. 351, pp. 1713-1720,
October 2000
© 2000 Oxford University Press
Original Papers |
Marking cell layers with spectinomycin provides a new tool for monitoring cell fate during leaf development
1 School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, UK
2 School of Biological Sciences, Manchester University, 3.614 Stopford Building, Oxford Road, Manchester M13 9PT, UK
Received 28 January 2000; Accepted 9 June 2000
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Abstract |
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Spectinomycin, an inhibitor of plastid protein synthesis, can be used to mark specific cell layers in the shoot meristem of Brassica napus. Pale yellow-green (YG) plants resulting from spectinomycin-treatment can be propagated indefinitely in vitro. Microscopic examination showed that YG-plants result from inactivation of plastids in the L2 and L3 layers and are composed of a pale green epidermis covering a white mesophyll layer. Epidermal cells of YG and normal green plants are similar and contain 10–20 small pale green plastids. YG plants are equivalent to periclinal chimeras with the important distinction that there is no genotypic difference between the white and green cell layers. Periclinal divisions of epidermal cells take place at all stages of leaf development to produce invaginations of green mesophyll located in sectors of widely varying sizes. A periclinal division rate of 1 in 3000–4000 anticlinal divisions for the adaxial epidermis, was 2–3-fold higher than that estimated for the abaxial epidermis. Analysis of white and green mesophyll showed that chloroplasts are essential for palisade cell differentiation and this requirement is cell-autonomous. Stable marking of cell lineages with spectinomycin is simple, rapid and reveals the requirement for functional plastids in cellular differentiation.
Key words: Brassica, cell lineage, epidermis, leaf development, spectinomycin.
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Introduction |
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The formation of organs from specific cell lineages is a key aspect of development. In transparent multicellular organisms with a relatively small number of cells, such as Caenorhabditis elegans, the developmental pathway of cells from zygote to adult can be defined by light microscopy (Sulston et al., 1983
). In more complex organisms, visual mutations, reporter genes, and cell-specific epitopes provide markers for monitoring cell fate. Other approaches used to mark cells include: (1) introduction of reporter genes into a subset of cells via retroviral vectors (Turner and Cepko, 1987), (2) use of cell type-specific promoters to express reporter genes (Malamy and Benfey, 1997), and (3) activation of pigment genes by recombination (Carpenter and Coen, 1995; Kilby et al., 1995). Cells can also be labelled by intracellular injection of fluorescent dyes (Harrison and Perrimon, 1993; Dale and Slack, 1987) or reporter enzymes (Weisblat and Stent, 1982). This simple approach obviates the need for mutation or molecular engineering. However, the extent to which cell fate can be followed is limited by cell division, which dilutes the marking reagent. Reagents that mark cells permanently without altering the genotype have not been described.
Chloroplasts have been used extensively to distinguish between cell-lineages for developmental studies in plants. Analyses of genetic mosaics composed of green and white cell layers have established the contribution of cells from the L1, L2 and L3 layers of the shoot apical meristem to the body of the plant, especially the leaves (Tilney-Bassett, 1986; Poethig, 1987). A method based on spectinomycin, an inhibitor of plastid protein synthesis, has been developed for permanently inactivating the developmental programme of chloroplasts from proplastids (Zubko and Day, 1998). Inactivation of plastid protein synthesis coupled with ribosome turnover and plastid divisions produces ribosome-deficient plastids that cannot express plastid genome-encoded proteins (including ribosomal proteins) and are permanently disabled. Green-white variegated plants, complete albinos and pale yellow-green (YG) plants are produced from Brassica napus seeds and buds exposed to spectinomycin.
Selective inactivation of plastids in one or more of the L1, L2 or L3 layers with spectinomycin would produce the plants schematically shown in Fig. 1. Inactivation of plastids in the leaf mesophyll, which normally contains the majority of chloroplasts would be the most easily identified. With this rationale YG plants isolated in a previous study were studied (Zubko and Day, 1998) in more detail. The analyses show that spectinomycin can be used to inactivate plastids irreversibly in specific layers of the shoot meristem.
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Materials and methods |
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Generation of yellow-green B. napus plants
Aseptic plant material treated with spectinomycin was cultured on MS medium (Murashige and Skoog, 1962
) containing 100 mg l-1 myo-inositol, 1 mg l-1 thiamine hydrochloride, 2 mg l-1 pyridoxine hydrochloride, 1 mg l-1 6-benzylamino-purine, 80 µg l-1 
-naphthalene-acetic acid, 30 g l-1 sucrose, 0.5 g l-1 2-(N-morpholino)-ethanesulphonic acid, and 1% w/v agar.
Leaves of Brassica napus with a distinctive yellow green (YG) phenotype were produced by three different methods: (i) seeds were soaked in spectinomycin solution (2–5 mg ml-1, Duchefa Biochemie) before being placed on spectinomycin-free media (Zubko and Day, 1998), (ii) seeds were germinated on medium containing 0.5 mg ml-1 spectinomycin (Zubko and Day, 1998) or (iii) vegetative shoots were cultured on solid medium and subsequently transferred to the same medium containing 0.2 mg ml-1 of spectinomycin dihydrochloride. Spectinomycin-induced bleaching was observed over the next 3 months. Bleached plants were then transferred onto the same medium but without the antibiotic. During the following 3–6 weeks of growth, plants exhibited extensive green-white variegation. Pure green or albino phenotypes segregated out during subsequent in vitro propagation (Zubko and Day, 1998). Amongst variegated plants the YG phenotype was easily detected as pale yellow-green leaves amongst green and albino leaves. YG plants were generated by all three spectinomycin treatment procedures detailed above. It was possible to maintain YG plants by cutting and transferring YG shoots deficient in green-sectors to new solid media. For rapid growth plants were grown in plastic Magenta jars (Sigma) containing 20 ml of liquid medium. The YG plant line described in this paper (Bn-IS-YG1) was generated by propagating shoots on spectinomycin-containing medium (method iii above). This line has been propagated for 3 years on spectinomycin-free media.
Periclinal division rate of epidermal cells
Periclinal division of epidermal cells is a relatively rare event and may be expected to follow a Poisson distribution since around 20–60% of leaf surfaces lack sectors. For 20 large leaves, 4 adaxial and 12 abaxial surfaces lack sectors. For 20 small leaves, 8 adaxial and 12 abaxial surfaces lack sectors. Whole green leaves (meristem sectors) were not used in the analysis.
If a equals the periclinal division frequency then (Luria and Delbruck, 1943):
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); Nt is the number of cells at time t; C the number of leaves scored (20 in this case).
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500 epidermal cells mm-2 were estimated and sectors of less than 0.04 mm2 (20 cells), which would not be visible by eye, were excluded. From the average leaf areas (Fig. 9A
), and only scoring sectors of 0.04 mm2 and above Nt can be estimated as 4200 (85 000/20) for large leaves and 1600 (32 000/20) for small leaves. This allows a to be calculated as 2.6-3.6x10-4 for adaxial periclinal divisions of large and small leaves, respectively. The corresponding figures for abaxial periclinal divisions are 8.5-16x10-5. The calculations based on the Luria–Delbruck equation assume the probability of periclinal divisions to be the same during leaf development. The estimates based on the adaxial surfaces of large and small leaves are in reasonable agreement. The 2-fold difference between the calculated abaxial periclinal divisions for large and small leaves is acceptable given the relatively small number of abaxial sectors which makes the calculations more sensitive to chance variations between samples. Fusion of adjacent independent sectors would result in a single sector scored and would lead to an underestimate of the actual periclinal division frequency. Given the low frequency of sectors per leaf, sector fusion is unlikely to introduce large errors into these calculations.
Microscopic analyses
Methods for cell fixation and separation, confocal imaging and leaf embedding and sectioning are as published previously (Kinsman and Pyke, 1998). Areas of green sectors in leaves were determined by image capture and subsequent measurement with a Nikon Lucia image analysis system (Kinsman and Pyke, 1998).
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Results |
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Green plastids are restricted to the epidermis of B. napus YG plants
B. napus YG plants are relatively stable and have been maintained in aseptic culture for more than 3 years. The leaves of YG plants are similar in shape to green leaves (Zubko and Day, 1998
) and are very unlike the elongated leaves of albino plants. A significant proportion of YG leaves has one or more dark green sectors of varying sizes (Fig. 2). The pale yellow-green phenotype contrasts sharply with sectors, which have the coloration of normal green leaves. Comparisons were made between the distribution of green plastids in YG plants and normal green plants, and between YG and green leaf areas located at sector boundaries.
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Confocal imaging by red autofluorescence from chlorophyll was used to compare the distribution of chloroplasts in green and YG leaves. Confocal optical sections through the adaxial (top) epidermis and through the mesophyll tissue revealed red chloroplast autofluorescence in the epidermis of both green (Ep-Gr2) and YG (Ep-YG1) leaves (Fig. 3A
, C). In contrast, Fig. 3B and D shows that chlorophyll autofluorescence was not detected in the mesophyll of YG plants (Mes-YG1), but was high in the mesophyll of green plants (Mes-Gr2). The abaxial (bottom) epidermis of YG and normal green plants also produced chloroplast autofluorescence (not shown). These data are consistent with the finding (Zubko and Day, 1998) that protoplasts prepared from whole leaves of YG plants revealed many chlorophyll-deficient cells and a reduced chlorophyll content in chlorophyll-containing cells. The chlorophyll-deficient cells were most likely to be mesophyll cells and those with reduced chlorophyll content were most probably epidermal cells. Figure 4 shows confocal imaging across the boundaries of two green sectors. Autofluorescence from the epidermis (Ep-YG) is continuous across both sector boundaries (Fig. 4A, C). In contrast, a discontinuity in chlorophyll autofluorescence was detected in the mesophyll layer across the sector boundaries (Fig. 4B, D). Bright autofluorescence was only detected in green areas.
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The results demonstrate that YG plants are deficient in green mesophyll and are composed of a green epidermis and white mesophyll. The YG phenotype arises because the chloroplasts in the epidermis of YG leaves are sufficient to impart a pale yellow-green colour to the leaf. Since the epidermis is derived from L1 whilst the mesophyll is formed from the L2 and L3 layers of the meristem, YG-plants result from inactivation of plastids in the L2 and L3 layers (Fig. 1A
). Green sectors arise from the restoration of functional green mesophyll in YG leaves.
Analysis of plastids in YG plants
Fixed isolated cells from YG leaves were examined by light microscopy. The irregular shaped epidermal cells from green and YG leaves clearly contain a small number of pale plastids (Fig. 5A, B). These are shown at higher magnification in Fig. 5C and D. Both adaxial and abaxial epidermis contains pale green chloroplasts in normal green and YG leaves. The mesophyll cells from green leaves contain a large number of green plastids (Fig. 5A) whilst those from YG plants are devoid of plastids (Fig. 5B). Observation of epidermal cells in YG and normal green leaves, respectively, shows that this cell type contains between 10 and 20 plastids per cell which are easily recognizable by light microscopy. These epidermal plastids do not appear to be abnormal in YG leaves. The sizes of epidermal and mesophyll plastids from YG plants, green sectors on YG plants and normal green leaves were compared (Fig. 6). In normal green plants, mesophyll plastids with a mean plan area of 29 µm2 are larger than epidermal plastids with a mean plan area of 12 µm2. Epidermal plastids from YG plants are larger 20 µm2 than those from normal green leaves. Epidermal plastids in green sectors from YG plants are also large showing continuity of epidermal characteristics across sector boundaries. The larger sizes of epidermal plastids in YG plants may reflect a regulatory mechanism to compensate for a lack of functional chloroplasts in the mesophyll.
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The mesophyll in green sectors of YG leaves and normal green leaves appeared indistinguishable. The relationship between chloroplast number per cell and mesophyll cell size was examined in detail and found to be the same in green sectors and normal green leaves (Fig. 7
). The chloroplast number varied from 20 to 100 per cell depending on cell size.
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) and mesophyll cells from green sectors on YG leaves (
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Green sectors result from periclinal divisions of epidermal cells during leaf development
Green sectors, which appear on YG leaves, contain a normal mesophyll (see above). These sectors vary greatly in size ranging from sectors which extend the entire length of the leaf and cover the majority of the lamina (Fig. 2C) to very small sectors which are composed of only a small number of mesophyll cells. Periclinal divisions in the epidermis are the most plausible explanation for the origin of green sectors and evidence to support this hypothesis comes from three distinct observations. Firstly, the pattern of green sectors on a YG leaf can differ on the adaxial and abaxial leaf surface (compare A and B in Fig. 8). Also note that examination of 20 small and large leaves revealed more sectors on the adaxial surface than the abaxial surface (Fig. 9B). These observations suggest that periclinal divisions can occur independently in either epidermis. Transverse sections show that green sectors always appear to originate from either the adaxial or abaxial epidermis and penetrate the leaf thickness to different depths (Fig. 8C). They have not been observed as solely internal tissues within the leaf thickness. Secondly, an analysis of the sizes of green sectors and their position on the lamina indicates that large sectors tend to originate from the leaf base and extend toward the leaf tip whereas small sectors tend to lie toward the top of the leaf (Fig. 10). The relationship between sector length, as a proportion of leaf length, and its position on the leaf shows a significant negative correlation. This would be expected if periclinal divisions in the epidermis occur at different times throughout leaf development such that the resulting sector reflects to some degree the timing of these divisions. The wide variation in sector sizes indicates that periclinal divisions of the epidermis must occur throughout leaf development from very early to late stages. This is also borne out by comparing the number of sectors on small and large leaves (Fig. 9B). Thirdly, in spectinomycin-induced fully albino lines of B. napus, in which all plastids in all cell types are ribosome deficient (Zubko and Day, 1998), including those in the epidermis (data not shown), green sectors have not been observed.
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Differentiation of palisade mesophyll cells requires functional chloroplasts
Dysfunctional plastids in the mesophyll layer of YG plants result from loss of plastid ribosomes due to spectinomycin (Zubko and Day, 1998) rather than mutation. Since YG and green leaf areas have the same genotype this allows the role of functional plastids in cellular differentiation to be assessed. The importance of chloroplasts to the proper development of the mesophyll is demonstrated by transverse sections across two sector boundaries from separate leaves of a YG plant (Fig. 11A, B). In areas where the leaf is YG and the mesophyll cells lack functional chloroplasts, the mesophyll tissue lacks a definite palisade mesophyll layer and all the mesophyll cells are generally of an isodiametric appearance. However, in green sectors, distinct palisade mesophyll cells can be observed below the adaxial epidermis and are recognisable by their characteristic elongate morphology (Fig. 11A, B). This demonstrates that chloroplasts are required for palisade mesophyll cell differentiation since in neighbouring cells lacking functional plastids typical elongate palisade mesophyll cells are not observed.
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Discussion |
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A method for permanently marking specific cell layers in plants has been developed. The procedure is based on the fact that spectinomycin inactivates plastid protein synthesis irreversibly, thereby creating clonal lines of white cells. White cells containing dysfunctional plastids can be distinguished from cells containing green plastids. Using this procedure B. napus YG plants, in which a pale green epidermis encloses tissues with dysfunctional white plastids, have been generated and studied. These plants are equivalent to periclinal chimeras with the important distinction that the green and white layers are genotypically identical in the spectinomycin-generated plants. YG plants contain an L1 layer that is marked by green plastids and can be maintained indefinitely by vegetative propagation in vitro. The dearth of L1 markers in Arabidopsis thaliana (Dolan and Roberts, 1995
), a related species subject to intense developmental biology research, makes these YG plants an especially valuable resource for studying the lineage of epidermal cells in Brassicaceae.
Spectinomycin-based marking of cell layers is simple and rapid. The treatment regime used here involved exposing plant material to a spectinomycin concentration below that used to generate wholly albino plants. Plants with pale green plastids in the epidermis and white plastids in the mesophyll cell layers appear pale yellow-green and are distinct from albino and normal green plants. YG plants were readily isolated as a distinct class of plants (Zubko and Day, 1998). It should also be possible to produce plants in which one or more of the other cell layers are marked with green plastids. Plants in which the epidermis is white and the mesophyll green would be harder to distinguish from normal green plants (Fig. 1). The efficiency of screening, accessibility of spectinomycin to plastids, possible resistance of certain plastid types to spectinomycin and viability of the resulting plants will all play a role in determining the variety of patterned plants that can be produced with spectinomycin.
YG plants are ideal for studying epidermal plastids which would normally be masked by the more abundant mesophyll chloroplasts. In B. napus, epidermal plastids are pale-green, small in number (10–20 plastids per cell) and are insufficient for phototrophic growth. YG plants must be grown on media containing sucrose. In general, epidermal plastids are normally small and contain lower levels of chlorophyll and photosynthetic protein components (Dupree et al., 1991) but there is variation between species. Whilst in B. napus (this work) and A. thaliana (Pyke and Leech, 1994) the numbers and sizes of epidermal plastids are small, in other species such as tobacco (Dupree et al., 1991) epidermal plastids can be well developed and of a similar size to mesophyll chloroplasts. If epidermal cells are less susceptible to spectinomycin this could be a consequence of reduced (1) plastid protein synthesis, (2) plastid ribosome turnover, (3) plastid divisions, (4) cell division in these cells relative to mesophyll cells. Cell and plastid divisions are important components of spectinomycin-based induction of ribosome-deficient plastids (Zubko and Day, 1998; unpublished results).
The presence of green sectors on YG leaves reflects periclinal division of green epidermal cells. Since the epidermal chloroplasts in YG leaves contain sufficient plastid ribosomes for the development of normal epidermal chloroplasts, differentiation of these epidermal cell derivatives in the underlying mesophyll allows the development of normal mesophyll cells with normal green chloroplasts, presumably with each containing a large population of ribosomes. Positional information rather than cell lineage appears to be responsible for determining cell type. In the course of leaf development in a normal green plant, such periclinal divisions may occur, but would not be easily observed since all the mesophyll is green. The presence of green sectors does not greatly influence the morphology of YG leaves, suggesting that periclinal division of epidermal cells is a normal process in leaf development.
The observation that sectors in the upper part of the leaf can be very small implies that the epidermal derivative was produced at a late stage of leaf development. Extensive variation in the size of sectors suggests that such divisions can occur at any point during the course of leaf development. Since occasional fully green leaves can be generated from YG shoots, such periclinal divisions must occur also in the shoot apical meristem or very early in leaf development. A lack of detailed knowledge on the patterns of cell division in the B. napus leaf makes interpretation of sector sizes in relation to leaf development difficult. Sector size is not only determined by the timing of epidermal periclinal divisions but also the rate at which the resulting green mesophyll cell divides. However, the distribution of sector sizes on these leaves would suggest a basic pattern of leaf development similar to that described in A. thaliana (Pyke et al., 1991).
Invasion of cell layers is not uncommon in plants and green flecks on a pale background have also been observed in periclinal chimeras of Pelargonium zonale and Epibolium hirsutum mesophyll (discussed in Kirk and Tilney-Bassett, 1978) which like YG plants contain a green epidermis and white mesophyll. The frequency of periclinal division of epidermal cells in tobacco leaves has been measured as 1 in 3100 divisions (Stewart and Burk, 1970). Our results based on B. napus plants grown in vitro are in reasonable agreement with the tobacco study. In B. napus a periclinal division frequency of 1 in 3000–4000 anticlinal divisions for adaxial epidermal cells and 1 in 6000–12000 anticlinal divisions for abaxial epidermal cells was estimated. These calculations based on the Luria–Delbruck equation (Luria and Delbruck, 1943) assume the probability of periclinal divisions to remain constant for each leaf surface during development. The difference in estimated periclinal division frequency between adaxial and abaxial epidermal cells hints at an asymmetry set in the leaf early in development.
Unlike periclinal chimeras and other genetic mosaics all the cells in YG plants have the same genotype. By comparing genetically identical cells with and without functional chloroplasts, it has been demonstrated that proper differentiation of palisade mesophyll cells with characteristic periclinal elongation only takes place in cells containing mesophyll chloroplasts. The possible role of chloroplasts in palisade development had been raised earlier from the analysis of plastid developmental mutants including pale cress in A. thaliana (Reiter et al., 1994), dag in Antirrhinum (Chatterjee et al., 1996) and dcl in tomato (Keddie et al., 1996). However, an equally plausible explanation in which these mutations prevent plastid development only as an indirect consequence of preventing palisade cell differentiation has not been ruled out. The analysis of palisade differentiation in YG plants, in which plastid dysfunction is not due to mutation, rules out this possibility. The sharp boundary between green and white mesophyll across green sectors in YG plants indicates that chloroplast mediation of palisade cell differentiation is highly cell-autonomous.
Most periclinal chimeras which have been exploited have arisen either by selection after random mutagenesis (Döring et al., 1999), exploitation of existing plant variants (Marcotrigiano and Morgan, 1988), grafting (Marcotrigiano and Bernatzky, 1995) or sorting out of green and mutant plastid types (Stewart and Burk, 1970). Marking cell layers with spectinomycin provides a new method for determining cell fate that is both simple and rapid. Cells are marked permanently and can be maintained indefinitely. In addition, the generation of dysfunctional plastids reveals the role of plastids in cellular differentiation. Unlike chimeras all cell layers in a treated plant have the same genotype. These advantages coupled with improvements in marking specific green cells, using techniques such by micro-injection, should make spectinomycin-based lineage tracking an attractive method for studying cell fate in plants.
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Acknowledgments |
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We thank Sarah Curtis for technical assistance and financial support from the BBSRC, INTAS-Ukraine and the Royal Society.
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Notes |
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3 To whom correspondence should be addressed at present address: Plant Science Division, School of Biosciences, University of Nottingham, University Park, Nottingham NG2 7RD, UK. Fax: +44 115 951 3298. E-mail: anil.day{at}man.ac.uk
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References |
|---|
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Carpenter R, Coen ES.1995. Transposon induced chimeras show that floricaula, a meristem identity gene, acts non-autonomously between cell layers. Development 121, 19–26.
Chatterjee M, Sparvoli S, Edmunds C, Garosi P, Findlay K, Martin C.1996. DAG, a gene required for chloroplast differentiation and palisade development in Antirrhinum majus. EMBO Journal 15, 4194–4207.
Dale L, Slack JMW.1987. Fate map for the 32 cell stage of Xenopus laevis. Development 88, 527–551.
Dolan L, Roberts K.1995. Two ways to skin a plant: the analysis of root and shoot epidermal development in Arabidopsis. Bioessays 17, 865–872.
Döring H-P, Lin J, Uhrig H, Salamini F.1999. Clonal analysis of the development of the barley (Hordeum vulgare L.) leaf using periclinal chlorophyll chimeras. Planta 207, 335–342.
Dupree P, Pwee K-H, Gray JC.1991. Expression of photosynthetic gene-promoter fusions in leaf epidermal cells of transgenic tobacco plants. The Plant Journal 1, 115–120.
Harrison DA, Perrimon N.1993. Simple and efficient generation of marked clones in Drosophila. Current Biology 3, 424–433.
Keddie JS, Carroll B, Jones JDG, Gruissem W.1996. The DCL gene of tomato is required for chloroplast development and palisade cell morphogenesis in leaves. EMBO Journal 15, 4208–4217.
Kilby NJ, Davies GJ, Snaith MR, Murray JAH.1995. FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis. The Plant Journal 8, 637–652.
Kinsman EA, Pyke KA.1998. Bundle sheath cells and cell-specific plastid development in Arabidopsis leaves. Development 125, 1815–1822.
Kirk JTO, Tilney-Bassett RAE.1978. The plastids. Amsterdam: Elsevier/North Holland Biomedical Press.
Luria SE, Delbruck M.1943. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 28, 491–511.
Malamy JE, Benfey PN.1997. Organization and cell differentiation in lateral roots of Arabidopsis thaliana. Development 119, 57–70.
Marcotrigiano M, Bernatzky R.1995. Arrangement of cell layers in the shoot apical meristem of periclinal chimeras influences cell fate. The Plant Journal 7, 193–202.
Marcotrigiano M, Morgan PA.1988. Chlorophyll-deficient cell lines which are genetically uncharacterized can be inappropriate for use as phenotypic markers in developmental studies. American Journal of Botany 75, 985–989.
Murashige T, Skoog F.1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–497.
Poethig RS.1987. Clonal analysis of cell lineage patterns in plant development. American Journal of Botany 74, 581–594.
Pyke KA, Leech RM.1994. A genetic analysis of chloroplast division in Arabidopsis thaliana. Plant Physiology 104, 201–207.
Pyke KA, Marrison JL, Leech RM.1991. Temporal and spatial development of the cells of the expanding first leaf of Arabidopsis thaliana (L.) Heynh. Journal of Experimental Botany 42, 1407–1416.
Reiter RS, Coomber SA, Bourett TM, Bartley GE, Scolnik PA.1994. Control of leaf and chloroplast development by the Arabidopsis gene pale cress. The Plant Cell 6, 1253–1264.
Stewart RN, Burk LG.1970. Independence of tissues derived from apical layers in ontogeny of the tobacco leaf and ovary. American Journal of Botany 57, 1010–1016.
Sulston JE, Schierenberg E, White JG, Thomson JN.1983. The embryonic cell lineage of the nematode Caenorhabditis elegans. Developmental Biology 100, 64–119.
Tilney-Bassett RAE.1986. Plant chimeras. London: Edward Arnold.
Turner DL, Cepko CL.1987. A common progenitor for neurons and glia perisists in rat retina late in development. Nature 328, 131–136.
Weisblat DA, Stent GS.1983. Cell lineage analysis by intracellular injection of marker substances. Current Topics in Development Biology 17, 1–31.
Zubko MK, Day A.1998. Stable albinism induced without mutagenesis: a model for ribosome free plastid inheritance. The Plant Journal 15, 265–271.
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