AN ANATOMICAL STUDY OF ROOT INITIATION IN STEM CUTTINGS OF HYBRID LARCH

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1 New Phytol (1978)81, AN ANATOMICAL STUDY OF ROOT INITIATION IN STEM CUTTINGS OF HYBRID LARCH By ALLAN JOHN Forestry Commission, Northern Researeh Station, Roslin, Midlothian EH25 9SY (Reeeived 4 January 1978) SUMMARY The stages in the development of roots in stem cuttings of Hybrid larch have been investigated. Callus develops from a new meristem after the establishment of a cortical vascular system. Roots arise from near the tip of this cortical vascular system and emerge as partially differentiated structures after approximately 42 days. INTRODUCTION The vegetative propagation of plants from stem cuttings has been used extensively in the horticultural industry for both herbaceous and woody species. The production of rooted stem cuttings of conifers has become important in recent years for producing plants for experimental purposes (Burdon and Shelbourne, 1974) and for commercial forestry (Kleinschmit, 1974). Vegetative propagation can be used to speed up the introduction of genetically superior stock produced from tree improvement programmes. Hybrid larch (Larix X eurolepis Henry), produced by crossing European larch {Larix deeidua Miller) with Japanese larch {L. kaempferi, Carriere) has proved difficult to produce in sufficient quantity from seed, especially as in many cases there are problems in matching the flowering times of the two species. Root development in stem cuttings can be sub-divided into three main stages (Hartmann and Kester, 1975). (1) Cellular differentiation and the initiation of groups of meristematic cells; (2) Differentiation of the cell groups into recognizable primordia; (3) Development and emergence of new roots. Carlson (1938) reported that latent root initials occurred in the stems of a number of species, e.g. Salix. Stem cuttings from these species are generally easy to root. Many species, however, do not contain latent root initials in the stem tissue and the formation of roots in stem cuttings of these species involves all three stages of root development. Latent root initials have not been described in the literature for any of the coniferous species commonly used in forestry (Dalgas, 1973). In conifers, especially those that are difficult to propagate, root primordia arise within basal callus tissue (Sato, 1956). The callus originates from cambial and phloem cells and is later enlarged by divisions of the cortical cells. Xylem elements differentiate within the callus in connection with the stem xylem. Haissig (1974) has hypothesized that root primordia are initiated from cells immediately adjacent to immature xylem present in the /78/ S Blackwell Scientific PubUcations 111

2 112 A.JOHN callus, i.e. vascularization of callus occurs prior to or concomitant with primordia initiation. If this hypothesis is true, roots are initiated only after a complex series of anatomical changes have occurred at the base of the stem cutting. Even though many studies have been made on the initiation of roots in the bases of stem cuttings, the entire process is not yet fully understood. A knowledge of the anatomical changes that take place in the cutting during the production of roots is necessary in order to define accurately the conditions to be used during the rooting process. The present study has been made to elucidate the anatomical changes that occur during the production of roots on stem cuttings of Hybrid larch. MATERIALS AND METHODS Four leafy stem cuttings, 20 cm long, were taken from each of 180 clones of 5-year-old Hybrid larch trees growing in Glentress Forest, South-East Scotland, on 13 August The cuttings were bundled, sprayed with water, placed in large polythene bags and stored overnight at 2 C. The basal 0.5 cm was trimmed from each cutting. The cuttings were then basally soaked for 2 h in either an aqueous solution containing 50 ppm indole-butyric acid as treatment or deionized water, as control. After soaking, the cuttings were struck into a mixture of coarse sand (2-5 mm) and sphagnum peat in a polythene greenhouse. Watering was carried out by a pressure mist system controlled by an electronic leaf. The cuttings were maintained under 18 h days with an air temperature of C and a propagation medium temperature of C. The experiment was designed with plots of twenty-four treated and twenty-four control cuttings randomized within each of fifteen blocks. Thirty treated and thirty control cuttings, two from each plot, were sampled at weekly intervals for 12 weeks. After visual assessment, the basal 1.0 cm of each cutting was trimmed and fixed in FAA, wax embedded, and serial sections prepared with a rotary microtome. The sections were stained with safranin and Fast Green, using methods modified from Jensen (1962). RESULTS (I) Macroscopic changes in cutting bases during rooting At the time of insertion, the cutting base was soft, with little lignification, green cortical tissues and a poorly developed periderm. After 7 days, the periderm and the tissues at the end had become browner. The basal 1 mm of the cutting seemed to be senescing after 14 days, with the phloem and outer tissues splitting from the central lignified xylem cylinder at the cambium. Swelling of the stem 1-2 mm from the base and splitting of the periderm were visible after 21 days. The swelling was more pronounced after 28 days and callus was developing towards the base of the cutting. Callus development reached a maximum after 35 days and protuberances appeared on its surface. The first roots emerged after 42 days. There was no difference between treated and control cuttings. The scheme outlined is the sequence of events deduced from macroscopic inspection of the cuttings that were rooting at the fastest rate. However, rooting in Hybrid larch is not synchronous and the emergence of the first root can occur over a period of days (John, 1977). The actual duration of the individual stages was different, but the developmental sequence remained the same.

3 Root initiation in larch 113 (2) Microscopic changes in cutting bases during rooting Plate 1, No. 1, shows the overall distribution of tissues at the cutting base before insertion into the propagation beds. The pith at the base of the trimmed stem was made up of rounded cells. Traces of primary xylem were present and the secondary xylem cell walls increased in thickness towards the cambium. The phloem sieve tubes (approximately square in tranverse section) were numerous and interspersed with associated cells. Medullary rays, averaging about twenty-four per transverse section, passed through the xylem, cambium and phloem. The cortical region, composed of relatively large cells with densely staining cytoplasm in some cases, varied in width from ten cells at the narrowest point to fifteen to twenty cells at the widest point, i.e. the leaf buttresses, and contained numerous air spaces and resin ducts. The initial periderm, formed below the epidermis was poorly developed and the phellem was composed of thin walled but collapsed cells. The first stages of the rooting process became apparent after 7 days. The tissues at the cut base, outside the secondary xylem, became separated from the secondary xylem in the region of the cambium and cell contents were not as heavily stained as previously (Plate 1, No. 2). There was proliferation of cells from the medullary rays into the cortical region, just above the senescing tissue. The cells were lightly staining, non-vacuolated and had obvious nuclei. Further separation of the outer tissues from the central zylem resulted in these cells becoming detached from the central medullary rays. This later separation was accompanied by the appearance in the cortex of groups of lightly staining ceils (Plate 1, No. 3). At a short distance from the cutting base, where the outer tissues were still in contact with the central xylem cylinder, divisions of the lightly staining cells were more radial and tangenital than at random. This resulted in the formation of a layer of cells near the inner region of the cortex (Plate 1, No. 4). The vascular cambium ceased normal function in this area, but remained active at a distance of 3 mm from the cutting base, though the newly formed xylem tracheids were shorter and had more pits than the tracheids of the original central xylem cylinder. The layer of cells formed towards the inner region of the cortex, in contact with the inner central xylem cylinder, differentiated into a new cambium and divided to form new cells towards both the inside and outside of the stem (Plate 2, No. 5). A layer of heavily staining parenchymatous cells was generally found between the central xylem and the newly formed cambium and associated cells. A row of cells developed into the cortical region by transverse division of the newly formed cambium after days (Plate 2, No. 6). The cambial cells in the cortex divided tangentially to form xylem initials towards the base of the cutting and phloem initials towards the apex. The xylem initials differentiated to produce short, heavily pitted tracheids (Plate 2, No. 7). The differentiation of recognizable phloem lagged behind that of the xylem initials. It was at this stage, days after insertion of the cuttings into the propagation beds, that callus formation commenced. Callus formed above and below the ring of intruding vascular tissue, and in the later stages of its formation, a distinct callus meristem was visible, though the origin of this meristem was unknown (Plate 2, No. 8). The development of the callus was accompanied by further development of the vascular strand formed from the new cambium. Tissue differentiaton within the vascular strand was limited mainly to the formation of new tracheids with little phloem formation occurring. In transverse section, the newly formed vascular tissues could be seen as a complete ring outside the central xylem cylinder, separated from it by a layer of heavily staining parenchymatous cells. Organized cell areas (tracheid nests) appeared at the basal tip of the newly formed vascular tissue within the callus after days, and it was from these areas that roots developed

4 114 A. JOHN (Plate 3, No. 9). The organized area rapidly differentiated into a protoroot,i.e. a meristem, that produced cells basipetally, and the new root emerged by extension of these cells approximately 42 days after cutting insertion. The main structure of the newly formed root at this stage of development was separated into two zones, an outer cortical region and a central stelar region containing no differentiated vascular tissues (Plate 3, No. 10). The differentiation of vascular tissues in the root occurred in a definite sequence. The callus vascular tissue forked at an early stage of root elongation and each half of the fork could be traced into the basal portion of the root (Plate 3, No. 11). The differentiation of xylem and phloem initials occurred in the extending root in the normal basipetal sequence. However, between the primary xylem of the extending root and the forked xylem at the base of the root, there was a region of undeveloped xylem and phloem initials (Plate 3, No. 12). The stem and root xylem became joined after further development of the xylem and phloem initials. Cambial acitivity in the root later gave rise to secondary xylem and phloem, enlarging the vascular connection between the developing root and the cutting base. It was found that treatment with indole-butyric acid did not modify any of the stages during the development of the cutting base. DISCUSSION Anatomical investigations of dynamic systems within plant tissues are only valid if thorough examinations of serial sections are made (Cameron and Thomson, 1969). Single sections, presented as photomicrographs, can be misleading. Those shown in this paper have been used to illustrate particular aspects of structure and organization and not as indisputable evidence of the developmental sequence of root formation. During this study, it was found that treatment of cutting bases with indole-butyric acid had no effect on either the rate of rooting or on the level of rooting achieved. Treatment with indole-butyric acid has been demonstrated to increase the level of rooting in cuttings from mature ortets, but not in those from young ortets (John, 1977). Macroscopic and microscopic investigation clearly show that there are three main stages of cellular differentiation in the development of roots in Hybrid larch cuttings. Firstly, there is the formation of a new cambium derived from cells of the medullary rays. This cortical cambium is continuous with the vascular cambium of the stem, but separated from the central xylem tissues in the basal region by parenchymatous cells. It divides rapidly to form a cortical vascular system that contains mainly xylem tracheids with little or no phloem development. The differentiation of recognizable phloem is not obvious until much later, often occurring after root emergence. During the second stage the development of the cortical vascular system is accompanied by callus formation. Callus tissue development usually follows the wounding of plant parts (Esau, 1964). However, the development of such tissue at the base of a Hybrid larch cutting is not just a wounding response but follows a sequence of other anatomical changes. The callus that developed to the lower side of the cortical vascular system was derived from a meristem, though the origin of this meristem is not clear. Callus formation in this region was similar to periderm formation i.e. a meristem (phellogen) divided to form living cells to the inside (phelloderm) cells to the outside that appeared to be senescing. The formation of callus to the upper side of the cortical vascular system was similar, though less pronounced in this region. Callus formation is a prerequisite of the third stage of the rooting process, because the

5 Root initiation in larch 115 roots arise from within the callus, near to the cortical vascular system. Smith and Thorpe (1975) traced the origins of root primordia in hypocotyl cuttings of Pinus radiata to single cells. Dalgas (1973), however, was able to trace primordia only to groups of cells in stem cuttings of Picea abies. Earlier, Cameron and Thomson (1969) suggested, because of their difficulty in finding the exact origin of root primordia in stem cuttings of P. radiata, that the actual groups of cells that initiate root primordia no longer exist by the time the primordia can be recognised. The first indication of primordia initiation in Hybrid larch cuttings was the appearance of ordered structures (tracheid nests) at the tip of the cortical vascular system. It must be assumed that the tracheid nests are associated with root formation, though their actual significance is not obvious, as the young root emerges with very little internal organisation. It has been suggested that functional vascular connections are required for the full development of the basal root system (Hoffmann and Kummerow, 1966). Root emergence and root elongation occur in Hybrid larch without complete vascularization. Vascular connection occurs by differentiation of vascular initials formed in the normal developmental sequence. The lack of vascular connection during the early stage of root emergence presents the propagator with a problem, since the transfer of water from the roots to the stem before vascular connection would be small. He cannot therefore lift the cuttings from the propagation bed and transfer them to less humid conditions until the roots have exceeded a minimum length. It is possible that changes in the temperature of the rooting medium, or changes in condition of the aerial environment may be required to speed up the rate of development of vascular tissues in the newly emerged root. ACKNOWLEDGMENTS I would like to thank Mrs C. Kinnaird for technical assistance and Drs M. P. Coutts and A. M. Fletcher for critically reviewing the manuscript. REFERENCES BURDON, R. D. & SHELBOURNE, C. J. A. (1974). The use of vegetative propagules for obtaining genetic information. A^.Z. /. For. Sci., 4, 418. CARLSON, M. C. (1938). The formation of nodal adventitious roots in Salix cordata. Am. J. Bot., 25, 721. CAMERON, R. J. & THOMSON, G. V. (1969). The vegetative propagation of Pinus radiata: Root initiation in cuttings. 5of. Gaz., 130, 242. DALGAS, K. F. (1973). Anatomical studies on cuttings of Norway spruce undergoing the rooting process. Forst Tree Improvement, 5, 1. ESAU, K. (1964). Plant Anatomy. John Wiley and Sons, New York. HAISSIG, B. E. (1974). Origins of adventitious roots. N.Z. J. For. Sci., 4, 299. HARTMANN, H. T. & KESTER, D. E. {1915). Plant propagation. Principles and Practices. Prentice HaU, New York. HOFFMAN, A. E. & KUMMEROW, J. (1966). Anatomische Beobachtungen zur Bewurzelung der Kartstreibe von Pinus radiata. Silvae Genetica, 15, 35. JENSEN, W. A. (1962). Botanical Histochemistry. W. H. Freeman, San Francisco. JOHN, A. (1977). Vegetative propagation of Hybrid larch {Larix X eurolepsis (Henry) in Scotland. In: Vegetative Propagation of Forest Trees, Physiology and Practice, pp Uppsala, Sweden. KLEINSCHMIT, J. (1974). A programme for large scale cutting propagation of Norway spruce. N.Z. J. For. Sci, 4, 359. SATO, S. (1956). Anatomical studies on the rooting of cuttings in coniferous species. Tokyo Univ. For. Bull., 51, Ul. SMITH, D. R. & THORPE, T. A. (1975). Root initiation in cuttings of Pinus radiata seedlings. 1: Developmental sequence./, exp. Bot., 26,184.

6 116 A.JOHN EXPLANATION OF PLATES PLATE 1 No. 1. T.S. of stem base before insertion into propagation bed. X 135. No. 2. L.S. of stem base with senescing basal cortex (SBC). X 135. No. 3. T.S., 1 mm from the base, witli lightly staining cells (CC) in separated cortical tissue. X 135. No. 4. T.S., 2 mm from the base, with lightly staining cells forming layers (L). X 135. PLATE 2 No. 5. T.S., 2 mm from the base, with the new cambium (NC) separated from the central xylem cylinder (CXC) by a layer of parenchymatous cells (PC). X 135. No. 6. L.S., 1 to 2 mm from the cutting base, with senescent basal tissue (SBT) and early development of the cortical cambium (C). X 135. No. 7. L.S. of cortical tissue with cells differentiating into short, pitted tracheids. X 300. No. 8. L.S. of tissues at the base with development of the cortical vascular system (CVS), formation of upper callus (IJC) and lower callus (LC) with the lower callus meristem (M). X 80. PLATE 3 No. 9. T.S. of lower basal tissue with a tracheid nest (TN) at the tip of the cortical vascular system. No. 10. L.S. of an emerging, partially differentiated root. X 45. No. 11. L.S. of basal tissues with the development of vascular tissue (VT) from the cortical vascular system into the base of an emerging root (R). X 50. No. 12. Diagrammatic L.S. through the base of a cutting during the early period of root elongation, approximately. X 40.

7 THE NEW PHYTOLOGIST, 81, 1 PLATE 1 T^ft:'k^-L<^.-4 4 iohn ROOT INITIATION IN LARCH (facing p. 116)

8 THE NEW PHYTOLOGIST, 81, 1 A. JOHN INITIATION IN LARCH PLATE 2

9 HE NEW PHYTOLOGIST, 81, 1 PLATE 3 CORTICAL VASCULAR SYSTEM ROOT VASCULAR INITIALS ROOT APEX iohn ROOT INITIATION IN LARCH CENTRAL XYLEM CYLINDER UPPER CALLUS LOWER CALLUS ROOT VASCULAR SYSTEM

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