Intrusive growth of primary and secondary phloem fibres in hemp stem determines fibre-bundle formation and structure

Abstract

Plant fibres-cells with important mechanical functions and a widely used raw material-are usually identified in microscopic sections only after reaching a significant length or after developing a thickened cell wall. We characterized the early developmental stages of hemp (Cannabis sativa) stem phloem fibres, both primary (originating from the procambium) and secondary (originating in the cambium), when they still had only a primary cell wall. We gave a major emphasis to the role of intrusive elongation, the specific type of plant cell growth by which fibres commonly attain large cell length. We could identify primary phloem fibres at a distance of only 1.2-1.5 mm from the shoot apical meristem when they grew symplastically with the surrounding tissues. Half a millimeter further downwards along the stem, fibres began their intrusive elongation, which led to a sharp increase in fibre numbers visible within the stem cross-sections. The intrusive elongation of primary phloem fibres was completed within the several distal centimetres of the growing stem, before the onset of their secondary cell wall formation. The formation of secondary phloem fibres started long after the beginning of secondary xylem formation. Our data indicate that only a small portion of the fusiform cambial initials (<10 %) give rise directly or via their derivatives to secondary phloem fibres. The key determinant of final bundle structure, both for primary and secondary phloem fibres, is intrusive growth. Through bi-directional elongation, fibres join other fibres initiated individually in other stem levels, thus forming the bundles. Our results provide the specific developmental basis for further biochemical and molecular-genetic studies of phloem fibre development in hemp, but may be applied to many other species.

Keywords: Cambium; Cannabis sativa; hemp; intrusive growth; phloem fibre bundles; plant fibres.

Figure 1.

Scheme of plant sample collection and number of primary and secondary phloem fibres in the cross-sections of hemp stems in various internodes in (A) 67-day-old and (B) 117-day-old plants. To the left, stem height (m) and 1 to 9—number of a stem internode. In the upper part of the stem with alternating phyllotaxis, fibres were counted at a distance 10 mm from the SAM for 67-day-old plants, which corresponds to the middle of the upper part of 117-day-old plants. *Significant difference determined by Student's t-test (P < 0.05) in number of primary phloem fibres in 67- and 117-day-old plants. Other than in the top part of the stem there are no statistically significant changes in primary phloem fibre numbers in any stem internode through the 50-day interval. Thus, the intrusive elongation of the primary phloem fibre is completed within the top part of growing hemp stem.

Figure 2.

Early stages of primary phloem fibre development in hemp. (A) A longitudinal almost median section of the apical part of the hemp stem showing (B) procambial cells, (C) primary phloem fibres at the stage of symplastic growth and (D) primary phloem fibres at the stage of intrusive growth. Procambial strands that can be identified at 0.4 mm from the SAM give rise to primary phloem fibres (pf). At a distance of 1.2–1.5 mm from the SAM, parenchyma cells (p) are found between the procambium (pc) and the fibres, confirming that the procambium had ceased to form primary fibres below this stem region. Primary phloem fibres at the stage of symplastic growth—narrow, elongated cells with at least two elongated nuclei (n)—still have flat ends (marked by arrowheads). Fibre ends become tapered when the intrusive growth of primary fibres starts. Arrows indicate position of B, C and D on A. Distance (mm) from the shoot apex is indicated in A. n, nuclei; ph, phloem; x, xylem. Bar: A = 200 µm, B–D = 10 µm.

Figure 3.

Cross-sections of the top part of a hemp stem, stained with Calcofluor White. (A and B) Primary phloem fibres at a distance of 10 mm from the SAM. The fibre bundles are being formed in the inner part of the cortex; parenchyma cells (p) and laticifers (l) are present between the fibres and the procambium. (C and D) Primary phloem fibres at a distance of 80 mm from the SAM. Bundles of primary phloem fibres (pf) are already formed meaning that the intrusive growth has ceased. Fibres located at the bundle side closest to the stem periphery are the first to start secondary cell wall deposition. e, epidermis; col, collenchyma; pc, procambium; pf*, primary phloem fibres with thickened cell wall; tr, trichome. Bar: A and C = 50 µm, B and D = 20 µm.

Figure 4.

Distribution of primary (grey) and secondary (black) phloem fibre length in mature hemp stem (frequency of abundance of different length fibres). Primary phloem fibres are significantly longer than secondary ones. n = 100.

Figure 5.

Cross-sections of a hemp stem, from the middle part of seventh internode stained with Calcofluor White. (A–C) Sections of 67-day-old plants. (A) A general view of the stem part; a white rectangle marks the area of secondary fibres and corresponds to C. (B) Stem area above A (B*—part of B in transmitted light channel); small groups of the intrusively growing secondary phloem fibres (marked by arrows) near the cambium. (C) Bundles of secondary phloem fibres with primary cell walls. (D) Secondary phloem fibres with thickened cell walls in 117-day-old plants at the same level as in B. The vascular cambium (c) in the hemp stems starts to produce secondary phloem fibres only at a distance of 600–700 mm from the SAM (seventh internode from the plant base). c, cambium; col, collenchyma; pf, primary phloem fibres; pf*, primary phloem fibres with thickened cell wall; sf, secondary phloem fibres; sf*, secondary phloem fibres with thickened cell wall. Bar: A = 100 µm, B–D = 20 µm.

Figure 6.

Serial cross-sections of a stem in the region of secondary phloem fibres, showing the cell wall thickness in the tip and middle part of one and the same fibre. Right column—larger magnification of the area marked on the left by a red rectangle. In the enlarged area shown in the right panel the fibre tip is indicated by an arrow. Phloem fibres have quite uniform thickness of the cell wall throughout their cell length. Therefore, fibre elongation and cell wall thickening do not coincide. pf, primary phloem fibres; sf, secondary phloem fibres. Distance between: A and B = 20 µm, B and C = 50 µm, C and D = 100 µm, D and E = 150 µm. Bar = 20 μm.

Figure 7.

Cross-section of a hemp stem from the middle part of the first internode of a 117-day-old plant stained with toluidine blue. Note the formation of dilatation meristems (dm) in two rays between the secondary phloem fibre bundles and the difference in diameters of primary and secondary phloem fibres. c, cambium; pf*, primary phloem fibres with thickened cell wall; sf*, secondary fibres with thickened cell wall; x, xylem. Bar = 50 µm.

Figure 8.

A scheme of the development of the secondary phloem fibre bundles demonstrating the major role of intrusive fibre growth. (A) Cambium region giving rise to secondary phloem elements (including secondary phloem fibres). Only a small portion of the fusiform cambial initials (one-seventh of the cambium circumference and two-third of the axial plane; marked in orange) give rise directly or via their derivatives to secondary phloem fibres. (B–D) Sequential stages of secondary fibre-bundle formation. Fibres in the bundle, which were formed by cambial initials located above and below that axial plane, are given in grey. Due to considerable increase in length by bi-directional intrusive elongation, fibres join other fibres initiated individually in other stem levels, thus forming the bundles and leading to the increase of fibre numbers on the cross-section. (D) Mature secondary phloem fibre bundle (fibres ceased intrusive growth and thickened cell wall).

Figure 9.

Structure of the (A) primary and (B) secondary phloem fibre bundles on the strips peeled off from the third internode of a 117-day-old plant hemp stem. Bundles frequently split and merge along the stem forming numerous anastomoses (marked by arrows). Bar = 100 µm.

Figure 10.

Scheme of the location of primary and secondary phloem fibres at different developmental stages within the stem of a 67-day-old hemp plant. For details, see text.