Clonal analysis of development in corn

In the corn plant the tassel, ear and each node develop from a small group of embryonal cells. Based on the analysis of clones, the shoot apex of the dormant embryo has been visualized to consist of 2-4 cells at the top set off to become the tassel, followed by 16 cells to become the 4-7 upper nodes (above the ear), and finally three levels of 32-cells-each representing the remaining nodes (Coe and Neuffer, 1978, pp. 113 ff. in The Clonal Basis of Development, eds. Subtelny and Sussex, Academic Press). The 32 cells at a given level develop into 2-3 nodes. The ear shoot develops from 2-4 cells which represent a subset of the 32 cells at that level.

The technique of marking cells through x-ray induced elimination of a dominant allele was used to investigate the elaboration of tassel, ear shoot and various nodes from embryonal cells. The ultimate aim is to arrive at a dynamic picture of corn plant development from the dormant embryo. Dry seeds heterozygous for color markers or mutations affecting the morphology of tassel and ear were x-rayed and grown, and sectors were scored in the mature plants.

Development of the tassel: Among over 3,000 plants examined (marked for one or more mutations, such as +/Vg, +/bz2, B Pl/b pl, +/Tu, +/Ts6, +/ra, R-r/r-g), 41 tassel sectors were observed. The majority of tassels (60%) developed from 4 ± 1 cells, the remainder from 2 to 14 cells. When the sector included the central spike, the latter was divided vertically into two halves showing that the subset of cells developing into the central spike comes from two separate cells. A vivid expression of this was observed in -/ra sectors where all the additional branch spikes were arranged only on one half of the central spike.

At the border of a sector, some of the male florets were also half sectored, but all the three anthers in such florets were either purple (R-r) or green (r-g). A single spikelet can develop from 2 cells but the stamens of a floret share a common cell. The mutations Vg, Tu, Ts6 and ra were found to be cell autonomous for expressivity in the tassel, and therefore their products must be cell limited. No sectors in tassel or ear were found among over 2,000 plants heterozygous for ts1, ts2, ts4, Tp1, Tp2, sk or D8.

Determination and development of nodes: The 1979 data on the apparent cell number (ACN) and the extent of sectors (Figs. 1 and 2) using a different strain of corn are similar to those reported by Coe and Neuffer (1978). The absolute ACN seems to depend on a particular strain, but invariably the nodes above the ear (nodes 15-20) show half the ACN of the lower nodes (nodes 8-15).
 
Node Level Dry Seed 2 Days 8 Days
21 10 - -
20 12 11 20
19 7 10 30
18 12 13 25
17 11 13 18
16 21 18 26
15 27 23 35
14 E 30 22 31
13 A 43 20 42
12 R 49 25 47
11 S 42 24 67
10 39 26 61
9 35 30 67
8 - 30 149
7 13 26 107

Figure 1. Apparent cell number at each node level for material x-rayed at dry seed stage and 2 and 8 days after sowing.

Figure 2.

Unlike some of the ferns, in the shoot apex of corn all cells divide. At the dormant embryo stage (except for the 2-4 cells destined to produce tassel) all the cells behave as independent components because the individual sectors extend 1, 2, 3, 4 or more nodes (Fig. 2). In a strict sense cells at the dormant embryo stage have not become destined to produce a particular node. As the development proceeds, the 32 meristematic cells at a level divide to produce nodal initials and another 32 cells representing the two remaining nodes. Each node initial cell is no longer an independent component and collectively the nodal initials become destined to produce a particular single node. The process of determination thus involves specifying the destiny of a group of cells to a specific node. In the plants x-rayed 8 days post-sowing (Fig. 3), the sectors extend for one node only between nodes 7 and 11 and 1.1 to 5 nodes at nodes 12 through 20. Groups of cells thus seem to become determined at successively higher levels and determination progresses from the base of the plant toward ear-bearing nodes. In the plants x-rayed 13 days post-sowing, determination is not only progressing at the base but is also initiated from nodes 15 toward the top (Fig. 3). This finding is consistent with the notion that at this time (13 days after sowing), the corn plant has been separated into two compartments, the upper one-third and the lower two-thirds.
 
Node Level Dry Seed 2 Days 8 Days  13 Days
20 6.0 7.6 5.0 4.0
19 4.3 4.3 3.3 -
18 1.0 2.5 4.5 -
17 4.0 3.5 3.0 2.0
16 1.7 2.5 2.1 1.0
15 2.4 2.3 2.0 1.0
14 E 2.2 5.3 1.5 1.5*
13 A 2.4 3.1 1.2 1.3
12 R 2.4 2.0 1.1 1.0
11 S 1.5 2.0 1.0 1.0
10 1.5 1.4 1.0 1.0
9 1.3 1.2 1.0 1.0
8 1.0 1.2 1.0 1.0
7 1.0 1.0 1.0 1.0

*Sector only in bract or husks

Fig. 3. Extent of sectors (in nodes) at each node level for material x-rayed at dry seed stage and 2, 8 and 13 days after sowing.

The majority of sectors induced at the dry seed stage started at the base of an internode, extended up through one or more nodes and terminated in a leaf. At the ear-bearing nodes, the sector started in the bract or husk and ended in the leaf situated one or two nodes above (Fig. 2). These results confirm the observations of Sharman (Ann. Bot. 6:245, 1942). The clonal analysis as well as the developmental anatomy both show that an internode corresponds to the lower half of a developing node, and that the ear shoot represents an axillary bud associated with the leaf above and not with the leaf in whose axil it appears.

Further development involves the formation of leaf and internode initials from nodal initials. While the leaf primordia are developing, the 32 internode initials divide leading to a widening of the axis: clones induced 8 days after sowing measured 1/64th at nodes 9 to 11 and 1/107 to 1/149th at nodes 7 and 8. In the plants x-rayed still later (13 days) the sectors at nodes 7-12 extended a single but entire internode. The internodes are therefore represented by a single layer of cells. Cell divisions leading to an increase of cell number in the vertical direction occur much later.

The results obtained thus far strongly suggest that the ground plan for all the nodes is laid down first while the shoot apex is still situated at the base of a corn seedling. The burst of growth occurring later mostly involves cell divisions and cell expansion in the internode initials.

Development of the ear shoot: Among over 6,000 ears examined, 138 ear sectors arising due to a loss of Bz2, P-WR, B, Pl or fertility factors were found. Only 28 of these entered the cob. An analysis shows that the ear shoot is derived from 4-6 cells which constitute a subset of 32 cells and, further, only two cell lineages extend and contribute to the cob and florets. The remaining two to four clones occur only in the husks and can terminate in any of the twelve or fourteen husks. Though the cob size in a given family is more or less constant, the relative contribution of two cells to the cob is highly variable. One cell can generate anywhere between 5 to 50% of cob and less frequently only a single cell extends into the cob. The extreme plasticity of the two cell lineages extending into the cob seems more or less a general rule and this feature can account for the unexpected ratio of green vs. white seedlings in a cross of half-white ij/+ female x +/+ male (see Rhoades, 1946, Cold Spring Harb. Symp. Quant. Biol. 11:202).

M. M. Johri* and E. H. Coe

*Permanent address: Molecular Biology Unit, Tata Institute of Fundamental Research, Bombay, India


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