AOBPreview originally published online on May 14, 2004
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Annals of Botany 94: 51-58, 2004
© 2004 Annals of Botany Company
Ficus rubiginosa ‘Variegata’, a Chlorophyll-deficient Chimera with Mosaic Patterns Created by Cell Divisions from the Outer Meristematic Layer
1 Plant Standards Branch, Department of Primary Industries, Private Bag 15, Ferntree Gully Delivery Centre, Victoria 3156, Australia and 2 Royal University of Veterinary and Agricultural Science, Bülowsvej 17, DK-1870 Frederiksberg C, Denmark
* For correspondence. Fax 61 03 92109396, e-mail david.beardsell{at}dpi.vic.gov.au
Received: 25 June 2003; Returned for revision: 5 November 2003; Accepted: 4 March 2004. Published electronically: 14 May 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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• Background and Aims Sections leaves of Ficus rubiginosa ‘Variegata’ show that it is a chimera with a chlorophyll deficiency in the second layer of the leaf meristem (GWG structure). Like other Ficus species, it has a multiseriate epidermis on the adaxial and abaxial sides of the leaf, formed by periclinal cell divisions as well as anticlinal divisions. The upper and lower laminae of the leaf often exhibit small dark and light green patches of tissue overlying internal leaf tissue.
• Methods The distribution of chlorophyll in transverse sections of typical leaves was determined by fluorescence microscopy.
• Key Results Patches of dark and light green tissue which arise in the otherwise colourless palisade and spongy mesophyll tissue in the entire leaf are due to further cell divisions arising from the bundle sheath which is associated with major vascular bundles or from the green multiseriate epidermis. Leaves produced in winter exhibit more patches of green tissue than leaves which expand in mid-summer. Many leaves produced in summer have no spotting and appear like a typical GWG chimera. There is a strong relationship between the number of patches on the adaxial side of leaves and the number on the abaxial side, showing that the cell division in upper and lower layers of leaves is strongly coordinated. In both winter and summer, there are fewer patches on the abaxial side of leaves compared with the adaxial side, indicating that periclinal and anticlinal cell divisions from the outer meristematic layer are less frequent in the lower layers of leaf tissue. Most of the patches are small (<1 mm in longest dimension) and thus the cell divisions which form them occur late in leaf development. Leaves which exhibit large patches generally have them on both sides of the leaves.
• Conclusion In this cultivar, the outer meristematic layer appears to form vascular bundle sheaths and associated internal leaf tissue in the entire leaf lamina.
Key words: Leaf chimera, leaf development, variegation, periclinal cell divisions, anticlinal cell divisions, mosaic, chorophyll-deficient mutant, Ficus rubiginosa, ornamental plant, vascular bundle, bundle sheath, sheath extension.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Ficus rubiginosa is a tree native to the central coast of New South Wales, Australia. A clone of this species with gold-edged leaves has been used as a landscape tree for many years in the eastern parts of Australia. Superficially it appears to be a periclinal chimera with a chlorophyll-deficient mutation in the second layer of the meristem (L2) (Derman, 1960; Tilney-Bassett, 1986; Beardsell and Considine, 1987). However, this cultivar differs from normal GWG chimeras (chlorophyll deficiency in the L2) because the entire leaf lamina including the golden leaf margin on both sides is spotted with irregular, sharply defined, small dark and pale green patches. These green patches overlay the green core of leaves on both the adaxial and abaxial sides. It is also an unstable chimera because unmanaged trees have a small number of branches with all green leaves.
Despite the large numbers of publications over the years on chorophyll-deficient chimeras including a number of reviews and books, there are few anatomical and developmental analyses of the large number of plant cultivars which exhibit mosaic chlorophyll deficiency patterns (Derman, 1960; Kirk and Tilney-Bassett, 1967; Tilney-Bassett, 1986; Rodermel, 2002). Kirk and Tilney-Bassett (1967) discuss mosaic plant chimeras at length, but make no hypotheses as to the cause of them. Several plant cultivars exist with chlorophyll-deficient mosaic patterns on leaves which are reminiscent of anthocyanin mosaics of the aleurone layer of maize caused by transposable elements. Structural and genetic analyses of these leaf mosaic chimeras have generally not been undertaken.
There are also few papers which provide evidence that the outer layer of the leaf meristem (L1) is involved in development of leaf tissue other than the epidermal layers. Renner and Voss (1942), Thielke (1948) and Bergann and Bergann (1983a) have shown that cells originating from L1 in various Pelargonium, Prunus and Peperomia cultivars can contribute to internal leaf tissue, but only near the leaf margin. Klekowski et al. (1996) have provided conflicting evidence that L1 forms internal leaf tissue in one clone of red mangrove.
The present paper investigates the unusual mosaic variegation patterns in leaves of a cultivar of Ficus rubiginosa by examining the leaf anatomy and distribution of chlorophyll in fresh leaf sections and in whole fully expanded leaves.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plant material
Representative leaves were collected from three trees of Ficus rubiginosa Desf. ex Pers. ‘Variegata’ growing in gardens in Melbourne, Australia.
Leaf appearance.
Leaves were collected at random from the periphery of tree canopies in August 2000 and February 2002 as representative of leaves which expanded in mid-winter and mid-summer. These were pressed for 2 d, photographed and photocopied to make a permanent record.
Leaf sections.
Unfixed, fresh frozen transverse sections approx. 6 µm thick were prepared from the green spotted gold margins and green centres of 80 leaves taken from the edge of the leaf canopy using the method of Beardsell and Considine (1987) but without pre-cooling with Freon. Chlorophyll distribution was determined using fluorescence microscopy as it is readily traced by its strong red fluorescence under blue light excitation (Beardsell and Considine, 1987).
Statistical analyses
Analyses of variance and regression analyses were done using Genstat 5 for Windows (Lawes Agricultural Trust, Rothamsted Experimental Station).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Leaf appearance
Most leaves of F. rubiginosa ‘Variegata’ which develop in summer have gold margins surrounding a pale green centre (Figs 1 and 2), typical of a stable periclinal chimera with the chlorophyll-deficient layer being derived from the L2 (Stewart, 1978). Both sides of entire leaves are often spotted with small patches of dark or pale green tissue which overlay the pale green core tissue (Figs 1 and 2). Although the number of patches <1 mm2 in area varies greatly in leaves that develop at any time, in winter there are approximately three times more produced on the adaxial side than in summer (Table 1). This ratio increases to 16 times when comparing the abaxial side in winter versus summer. Patches >1 mm in their longest dimension are also more frequent in winter, with ten times more on the adaxial side and 30 times more on the abaxial side than in summer.
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In winter, there are approximately twice as many patches on the abaxial side as on the adaxial side (Table 1). Despite great variation between individual leaves, the number of patches on the abaxial (Y) and adaxial (X) sides of leaves is strongly related, with 87·9 % of the variance accounted for by the relationship Y = –25·8 + 1·024X – 0·001062X2 during winter and the relationship Y = –20·6 + 0·54X (r2 = 82·3) when the winter and summer data are pooled (Fig. 3). In summer, the ratio of patches on the adaxial side increases to between five and eight times the number on the abaxial side. In both seasons and on both sides of leaves, there are between five and 12 times more patches less than 1 mm2 than those greater in area.
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Large sectors or patches which extend from the midrib to the leaf margin are much less frequent, with approximately only one in 100 shoots having such leaves (Figs 4 and 5). This reversion can take place on whole sides of leaves, or as sectors running from the midrib to the margin. This can occur anywhere from the tip to the base of the leaf, although the sectors occur more frequently near the base of leaves. In 16 leaves exhibiting such large sectors, both sides of the leaf were both affected, and the large sector on the adaxial surface aligned completely or partially with the large sector on the abaxial surface in 14 of these leaves. Very few leaves occur with sectors larger than 1 cm2 on one side only. The margins of large sectors are generally aligned at a more acute angle (mean approx. 40°) to the midrib than the angle between the primary veins and mid-vein (mean approx. 60°) (Figs 4 and 5). Clonal sectors in tobacco leaves are inclined at a similar angle to these sectors in Ficus rubiginosa (Poethig and Sussex, 1985). However, the general direction of the border of these sectors often deflects to follow the primary veins for some length before resuming a more acute alignment once again (Figs 4 and 5). The margins of these larger sectors are always delimited by secondary veins or primary veins.
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Figure 6 shows the abaxial side of a leaf near the border of the green centre and gold margin. The vascular system consists of a prominent mid-vein, and primary veins which extend from the midrib to form a network extending almost to the leaf margin. In leaf tissue between these primary veins is a network of secondary veins which form rings approx. 0·5 mm in diameter. Patches of green tissue occur over the entire leaf lamina and overlie the green centre. All green patches are bordered by vascular bundles on at least one side (Fig. 6).
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Leaf structure and chlorophyll distribution
A section taken in the green centre of a gold-edged leaf is shown in Fig. 7. Both the adaxial epidermis and abaxial epidermis are multiseriate with three or four cell layers, typical of the genus Ficus (Esau, 1958), although the inner layer of the adaxial epidermis could be a hypodermis as in Lophostemon confertus (Beardsell and Considine, 1987). The outer layer of the abaxial epidermis contains sunken stomata with strongly fluorescing chloroplasts in the guard cells. In the section shown in Fig. 7, the upper layer of palisade (P1) and the lower layers of spongy mesophyll tissue (S1) are devoid of chlorophyll, whereas the inner layers of palisade (P2) and spongy mesophyll (S2) tissue contain strongly fluorescing chloroplasts.
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Figures 8 and 9 show that patches of green tissue in the otherwise achlorophyllous margin are the result of groups of cells containing chlorophyll in various layers of palisade or spongy mesophyll tissue adjacent to vascular bundle sheaths or their extensions. In Fig. 8, a dark green patch of chlorophyllous cells in the upper palisade layer (P1) is adjacent to both the bundle sheath extension and inner layer of the multi-seriate epidermis. Another chlorophyllous patch (P2) in the lower layer of palisade cells underlies achlorophyllous palisade tissue and is also contiguous with the bundle sheath. Two other patches occur in the achlorophyllous lower spongy mesophyll tissue. Figure 9 shows patches of chlorophyllous tissue adjacent to the bundle sheath in the outer palisade layer (P1) and in the inner mesophyll tissue (S2). The bundle sheaths in Ficus rubiginosa extend to the inner layers of both the adaxial and the abaxial epidermis (Fig. 8).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The multiseriate epidermis of F. rubiginosa ‘Variegata’ must be formed by periclinal cell divisions from the outer layer of the leaf meristem in addition to the anticlinal divisions which normally form the epidermis of plant leaves (Foster, 1936; Esau, 1958). This is a different pattern of development to the formation of the hypodermal layers in plants such as Lophostemon confertus which take place mainly by anticlinal cell divisions from the L2 (Bergann and Bergann, 1983b; Beardsell and Considine, 1987). The colourless (except for green patches) outer palisade and spongy mesophyll tissue in F. rubiginosa ‘Variegata’ must be derived from the L2 as in other GWG (Green/White/Green) chimeras which have gold-edged leaves (Stewart, 1978; Tilney-Bassett, 1986; Beardsell and Considine, 1987). Similarly, the green core of the leaf which consists of the lower layer of palisade and inner layers of spongy mesophyll is derived from the third layer of the meristem (L3) as is usual in dicots including Pelargonium zonale (Foster, 1936; Stewart, 1978), Hedera sp., Ilex sp. and Euphorbia pulcherimma (Stewart, 1978) and Tristaniopsis laurina (Beardsell and Considine, 1987). The amount of green core tissue, presumably derived from L3 varies greatly in this cultivar, thus achlorophyllous tissue derived from L2 may displace L3 as occurs in most other dicots (Stewart and Derman, 1975; Tilney-Bassett, 1986).
The main question to be answered is the origin of the dark and light green patches and sectors which occur in the gold leaf margin and which overlie the green leaf centre. It is possible that the L2 in F. rubiginosa ‘Variegata’ contains an unstable mutation which reverts back to normal green. We have sectioned approx. 80 leaves, and both the epidermis and the inner core (L3 derived) are green with no evidence of a mosaic structure. A more likely explanation is that these patches are formed by anticlinal cell divisions from the vascular bundle sheaths which are genetically green and derived by periclinal division from the multiseriate epidermis (Fig. 10). The patches overlie both achlorophyllous and chlorophyllous parts of the leaf lamina (Figs 1 and 2). They could also occur by additional periclinal divisions from the multiseriate epidermis. Since the epidermal layer is formed by a mixture of periclinal and anticlinal divisions in Ficus spp. either of these two pathways is possible. Similar periclinal divisions occur from the hypodermis on the adaxial side of the leaf in Lophostemon confertus forming the sheath surrounding vascular bundles (Beardsell and Considine, 1987). Since both the pale and dark green patches and sectors are always bordered on at least one side by vascular tissue, the bundle sheath appears to be the more likely progenitor of these green patches. The occasional presence of small patches of inner spongy mesophyll tissue containing chlorophyll next to vascular bundles in the achlorophyllous margin also supports this origin. The question arises as to whether all bundle sheaths across the entire leaf lamina in Ficus rubiginosa are derived from L1. It is believed that, since the spots are only associated with vascular bundles and there are no small groups of chlorophyll-containing cells in the mesophyll separated from the vascular bundles, it is unlikely that they are the result of the release of an epigenetic silencing mechanism or a transposon insertion both of which could cause reversion from white back to green.
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Current understanding of leaf formation concludes that vascular bundles and are generally derived from the tissue in which they are embedded, namely L2 in monocots (Langdale et al., 1989). In the C4 monocot maize, ontogenetic studies of the formation of bundle sheaths indicate they are derived from the surrounding ground tissue (Bosabalidis et al., 1994); however, in barley, a C3 monocot, the bundle sheath appears to develop from the sub-epidermal layer. The only ontogenetic study of the origin of bundle sheath cells in dicots has been done on Atriplex rosea, a C4 species where the bundle sheath also appears to arise from the ground tissue in which it is embedded (Liu and Dengler, 1994). This species, however, does not have bundle sheath extensions which connect directly to the epidermal layers. Beardsell and Considine (1987) have shown that in Lophostemon confertus, the bundle sheaths and their extensions to the hypodermal layers in the centre of the leaf are formed from L2 displacing surrounding tissue derived from L3. There is, however, no previous literature showing that tissue of bundle sheaths in dicots may be derived from L1, although Renner and Voss (1942), Thielke (1948), Stewart (1978); Tilney-Bassett (1986) and Dawe and Freeling (1991) recognized that cells from L1 occasionally enter the L2 after periclinal cell division. The origin of vascular tissue in plants with bundle sheath extensions has not been closely examined in dicots since the survey of Wylie (1952), and only detailed developmental studies will determine if L1 contributes to their formation. The present paper, however, indicates that at least in Ficus rubiginosa, which has a multi-seriate epidermis and bundle sheath extensions, L1 appears to be the progenitor of vascular bundle sheaths in leaf mesophyll tissue which is otherwise derived from L2 or L3 (Fig. 10). Ontogenetic studies are needed to confirm if vascular bundle sheaths which extend from adaxial to abaxial epidermal layers in some dicot species can, under certain conditions, be derived from cells by organized displacement of L2 or L3 tissue by cells originating from L1 as indicated in Ficus rubiginosa ‘Variegata’. Since Beardsell and Considine (1987) have also shown that L2-derived cells may displace L3-derived mesophyll in bundle sheaths in Lophostemon confertus, the elegant model of leaf formation proposed by Poethig and Sussex (1985) and Poethig (1987) may not always operate in dicots which have bundle sheaths which extend from epidermal or hypodermal layers.
Klekowski et al. (1996) have indicated that L1 forms part of the mesophyll of leaves in a clone of red mangrove. These authors concluded that in red mangrove, L1 forms a multi-seriate epidermis and additional periclinal cell divisions form mesophyll tissue in the leaf margin. They base this conclusion on the achlorophyllous guard cell chloroplasts in one chimeral selection and the production of albino seedlings from fruits on variegated shoots. Figures in their paper indicate that this plant has a three-layered epidermis overlaying a double-layered hypodermis. The red mangrove with white leaf margins in Klekowski et al. (1996) probably has the GWG structure rather than the WGG (White/Green/Green) that they described; L2 forming the hypodermis and mesophyll tissue in the leaf margin as occurs in other dicots described in the literature (Stewart, 1978). The albino seedlings in red mangrove, assuming maternal inheritance of plastids, would thus be formed from a megaspore mother cell derived from L2 as is normal in dicots (Marcotrigiano and Bernatzky, 1995). Rhizophora spp. including red mangrove have a crassinucellar ovule (Davis, 1966) whereby the megaspore mother cell arises in a sub-epidermal layer of the ovule which would preclude maternal origin from L1. In addition, given the importance of functional chloroplasts in leaf stomatal guard cells (Zeiger et al., 1987; Quinones et al., 1996), it is questionable that L1 can support mutations in this histogenic layer if they result in greatly impaired photosynthetic function.
It is unclear why there are always more patches of chlorophyll containing tissue in the upper layers rather than the lower layers of mesophyll in Ficus rubiginosa, both epidermises having four layers of tissue, and thus a similar propensity for organized periclinal cell divisions from L1. Although the number of patches visible on the abaxial side of leaves is always less than on the adaxial side, the relationship between them is very strong. This shows that the cell division in the upper and lower layers of mesophyll tissue is strongly coordinated as indicated by Poethig (1997). This relationship also reinforces the hypothesis that the patches are due to regulated periclinal cell divisions and not due to random sorting out of different lines of cells or to genetic control of a transposable element system. The greater number of patches overlying the green core of leaves of this periclinal chimera in winter compared with summer could be due to changes in cell division patterns directed by any of the environmental changes which vary between these seasons in southern Australia. It is beyond the scope of the present project to determine whether the key factor is temperature regime, day length or light intensity or a combined effect of these factors. However, leaves expanding in summer have greater variation in the number of patches (Table 1) which indicates that temperature effects may be the dominant factor as this varies more than day length and light intensity.
If leaf development in Ficus rubiginosa is similar to that of tobacco, and since most of the patches are <1 mm2 in area, most of the cell displacement events from L1 into L2 and L3 occur after development of the leaf lamina (Poethig and Sussex, 1985; Poethig, 1987). Cell displacement events which result in a sector which extends from the midrib to the margin occur at two orders of magnitude lower frequency than the small patches. If the model of Poethig and Sussex (1987) holds for Ficus rubiginosa, these rare early displacement events presumably occur prior to the formation of the leaf lamina.
Since most leaves examined that had sectors of chlorophyllous tissue stretching from the midrib to the margin on the adaxial surface also had a similar sector on the abaxial side (Figs 4 and 5), these reversion events are the result of periclinal and anticlinal cell divisions triggered in both the upper and lower parts of mesophyll tissue early in leaf development. Since the sectors on the respective sides of the leaf are also generally not aligned, the cause is not the result of the same cell displacement event. Cell displacement of L2 from L1 very early in leaf development also results in the reversion to all green leaves in approximately one in 100 GWG shoots.
Since there are more periclinal cell divisions in the margins of leaves (Tilney-Bassett, 1986) it is not surprising that a disproportionate percentage of patches occur abutting the leaf margin (Table 1).
It is interesting to note that Werbrouck et al. (1997) have found that the fungicides prochloraz and imazalil cause similar leaf spotting and reversion to green in the golden leaf margin of GWG and GWW (Green/White/White) chimeras of Ficus elastica and F. benjamina grown in tissue culture. Werbrouck et al. (1997) concluded that this response is due to enhanced periclinal cell divisions from the green L1-derived tissue, and it presumably happens via a similar mechanism to that described in the present paper.
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ACKNOWLEDGEMENTS |
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This project was funded by the Department of Primary Industries (Victoria). Elizabeth James and Gowri Maheswaran critically reviewed the text. Pierre Debergh allowed us to review some of his group’s unpublished work. Peter Franz provided statistical advice, Andrew Hamilton prepared graphs and Publishing Solutions assisted with scanning and labelling figures. This paper is dedicated to the late Professor R. Bruce Knox, a great biologist, mentor and friend.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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