Proc. Indian Acad. Sci., Vol. 81 B, No. 3, 1975, pp. 101-110

Anatomy of the mature embryo and seedling of Picea smithiana (Wall) Boiss*

K. VENKATARATNAM,B. CHACKO, B. D. DESHPANDE AND S. K. PILLAI Department of Biological Sciences, Birla Institute of Technology and Science, Pilani 333031 MS received 27 September 1973 (Communicated by Prof. V. Puri, F.A.Sc.)

ABSTRACT The anatomy of the mature embryo and M-day old seedling of Picea smithiana (Wall) Boiss. has been described. The organization of the shoot and root apices in both is similar. The shoot apex shows five cytohistological zones, where the entire surface layer shows periclina[ divisions and a small subapical initials zone. In the radicular and root apices there is a common initiating zone for the stele and columella in the centre surrounded by another common initiating zone for the cortex and peripheral region of the root cap. In the mature embryo the kappe divisions of the protoderm resulting in the peripheral region of the cap can be noted, which, however, disappear in the root of the seedling. The 34-day old seedling shows a root-hypocotyl-cotyledon vasculature without any connection with the shoot, where no vasculature ha~ yet developed.

INTRODUCTION REPORTS on the structure of the mature embryo and seedling of gymnosperms are rare (Buchholz and Old 1933; Schopf 1943; Alien 1947 a, b; Tepper 1963, 1964; Gregory and Romberger 1972; Riding 1972). The shoot apex of the adult of Picea smithiana has been studied by Shah and Thulasi (1967). This report presents information about the anatomy of the mature embryo and 34-day old seedling of Picea smithiana.

MATERIALS AND METHODS Seeds of Picea smithiana (kindly supplied by Prof. R. V. Singh, Depart- ment of , Himachal Pradesh University, Solan, Simla Hills) were soaked in water for an hour, the seedcoat removed and the embryo fixed. Seeds germinated only after a cold treatment at about 5°C for 25 days or

* This work has boon financed in part by a grant made by the United States Department of Agriculture, Agricultural Re~mrch Servioz, authoris~d by Public Law 480. 10I B 1--March 75 102 K. VENKATARATNAM et al. more. The treated seeds were sown in the soil in September 1972, and they germinated in about 15 days. All materials were fixed in formalin-acetic acid-alcohol, processed through the alcohol-xylol series and embedded in paraffin. Serial sections, transverse and longitudinal, of the mature embryo and shoot and root apices of the seedlings, were cut at 8 ~, thickness. Trans- verse sections of the seedlings were taken at 15/~ thickness for studying the root-shoot transition. Northan's variation of Foster's schedule (Johansen 1940) for tannic acid-iron chloride and safranin was used in most cases. Chlorazol Black E also gave good results.

OBSERVATIONS The shoot apex of the mature embryo is dome-shaped initially (figures ] and 22), and conical after soaking in water (figure 2). The shoot apex of the 34-day old seedling, though bigger, exhibits the same shape, and the first needle primordia become evident (figures 3 and 23). The average height and width of the shoot apex of the mature embryo are 91.32~ and 205.02ft and of the seedling 139 ~ and 250-2 f~ respectively. Tanniferous contents are noticed in the seedling apices. In the embryonic apex many granular contents are noticed in the cells, which disappear with germination and advancement of growth (figure 22). Zonation of tke skoot apex--The following zones are evident. Zone 1. The apical initials--These cells occupy the summit of the apex and in the mature embryo range from 6 to 8 (figure 22). In the 34-day old seedling apex their number is only 4 or 5 (figures 23). Shah and Thulasi (1967) have reported 2 to 6 apical initials in the adult plant apices. The apical initials are large with spherical, deeply staining nuclei and cell contents. In the cells of the mature embryo the nuclei are more prominent and occupy three-quarters of the cell volume. The cell walls are more or less uniformly thickened and the corner thickenings reported by Shah and Thulasi (1967) were not observed. Both anticlinal and periclinal divisions occur with the former predominating (figures 22). Periclinal divisions in the apical initials contribute to the subapical initials proximally, the distal derivatives persisting as the apical initials. Periclinal divisions in the shoot apical cells of the young embryo at cotyledonary initiation was reported by Gregory and Romberger (1972) in . Periclines have been reported in the apical cells of the mature shoot of Torreya ealifornica by Kemp (1943), four species of Cupressaceae by AI Sheriff (1952), five species of by Jackman (1960), Cephalotaxus drupaeea by Singh (196I), Cupressus species by Pillai (1963 a), Thuja oriental& Thuja eompaeta, Juni- pcrus chinensis and Callitris robusta by Pillai (A. 1963 a). Anatomy of the mature embryo .. 103

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@ ab @ 6.,°.,° J . FOR I)2)3,S)2) )oo~ FOR 7-9 Figures 1-21. 1. Median L.s. of shoot apex of dormant mature embryo. 2. Median L.s, of shoot apex of embryo 1 hr after seed-wetting. 3. Median L.s. of shoot apex of 34-day old seedling. 4. Three-dimensional picture of seedling showing vasculature. 5. K6rper divisions in pecipheral region of root cap. 6. Median L.s. of radicle of mature embryo. 7. Median L.s. of root of 34--day old seedling. 8. Transection of root 2,050~ above root initials. 9. Transec- tion of root 4,500~ above root initials. 10. Transection of root 59,900p above root initials, 11. T.s. of hypoeotyl. 12. T.s. of hypocotyl 825 p below the shoot apex. 13. T.s. of hypocotyl 555~ below the shoot apex. 14. T.s. of hypocotyl 465p below the shoot apex. 15. T.s. of hypocotyl 375 p below the shoot apex. 16. T.s. of cotyledonary node. 17. T.s. of hypocotyl 225p below the shoot apex. 18. T.s. of hypocotyl 120p below the shoot apex. 19. T.s. of hypocotyl 80~ below the shoot apex. 20. T.s. of hypocotyl 30p below the shoot apex. 21. Vascular bundle in the cotyledon. i04 K. VENKATARATNAM et a~.

Zone 2. The surface layer on the flanks--This zone arises by anticlinal divisions of the apical initials and is not treated as part of the flanking zone. In the mature embryo this zone has elongated cells with small nuclei. In the embryo and seedling apices periclinal divisions are common (figures 22 and 23), the inner derivatives contributing to the flanking zone. Increased frequency of periclines is observed during the initiation of needle primordia, the cells becoming wider at the locus of initiation (figures 3 and 23). Similar periclines are also reported in Abiesconcolor, Piceaexcelsaand Pinus montana (Korody 1937). Zone 3. The sub-apical initials--This zone, comprised of the inner derivatives of periclines in the apical initials (figures 1, 2, 3, 22 and 23), has cells with prominent, deeply stained nuclei and lightly stained cell contents. There are no corner wall thickenings. As the seedling grows, the cells enlarge (figure 23). Gregory and Romberger (1972) reported variation in the volume of this zone during growth up to 148 days in Picea abies. They also reported vacuolation in the precotyledonary shoot apex. Periclines in the peripheral cells of this zone form the flanking zone and anticlinal divisions in the basal cells, the rib meristem. Zone 4. The rib meristem--The highly vacuolated cells elongated parallel to the long axis of the shoot are arranged in regular vertical rows. The number of rows depends on the number of the subapical initials present. The width of this zone increases basipetally by periclinal divisions followed by cell enlargement and this is evident during seedling growth also. Anticlinal divisions also occur. Pith mother cells are absent, as reported by Pillai (1964) in Araucaria spp. In the shoot apex of the embryo the maturation of pith is nearer to the apical initials. The formation of pith is a gradual process as in the case of Sequoia (Cross 1943), Araucaria spp. (Pillai 1964). As the rib meristem extends into the hypocotyl, the vacuolation and length of the cells increase (figure 3). Zone 5. The flanking Zone or peripheral rneristem--This zone is a combined product of the periclinal divisions of the surface layer on the flanks and the anticlinal divisions of the subapical initials. The cells are longer than broad and densely cytoplasmic. Distally this zone is very narrow but becomes wider due to subsequent T-divisions. During the initiation of needle primordia divisions occur in all planes. The flanking zone forms a cylinder around the rib meristem and pith and is the site of development of procambium, cortex of the axis, and the subepidermal tissues of the needle. Root apex--The structure of the root apex in the mature embryo and seedling is fundamentally the same (figures 6, 7, 24, 25). In the mature embryo a disc-shaped group of 5 to 6 initials is present, while in the seedling root apices, a transverse plate of about 4 ceils form the initials Anatomy of the mature embryo .... 105

(figures 24, 25). These initials produce the stele and pith proximally and columella distally. The radicular stele is broader than the stele of the seedling root, evidently due to frequent kOrper divisions (figures 24, 25). In the radicle of the mature embryo the deeply stained narrow cells of the stele can be easily distinguished from those of the cortex and also by the colourless endodermis separating the stele from the cortex (figure 24). In the radicle of the embryo the central cells of the stele mature into the pith a short distance proximal to the initials while in the seedling root apices the pith does not mature so closely (figure 25). In the seedling root apices the columella is uniformly 4 cells wide while in the mature embryo the columella is wider (figure 24). The meristematic tissue around the initials cuts off the cortex proximally and the cap distally. The outer cortical cells extend up to the tip of the root cap and surround the columella. Kappe divisions occur in the pericolumnar region (figures 24, 25). The cortical initials are less in volume compared with the stelar initials. These cells are longer than broad (figures 24, 25). But, they show similar affinity for stains. The peripheral region of the root cap is easily distinguished from the columella by the kappe type of cell arrangement. Besides this, they differ in their avidity for stains. There is no true epidermis, the function of the protoderm being served by the underlying layer of the cortex as the outer layers peel off. Tanni- ferous contents are present in the cells of the peripheral part of the seedling root and absent in those of the mature embryo (figures 24, 25). There is no clear demarcation between the cortex and the peripheral part of the root cap. The inner cortical cells give rise to the endodermis composed of colourless cells far to the proximal side of the root initials (figure 24). The '+ secretory cells," the initials of the tanniferous cells, are distri- buted irregalarly in the stele. Still higher up the vascular initials are arranged in groups with deeply stained nuclei. Structure of the seedling--Germination is epigeal, during which the hypocotyl along with the cotyledons still enclosed in the seedcoat, emerge out. The hypocotyl attains a height of 2" under laboratory conditions whereas it is shorter in the seeds germinated in the garden. The cotyledons unfold only after the hypocotyl ceases elongation. The entire process of germination up to the dropping of the seedcoat takes nearly forty days after seed wetting. The number of cotyledons ranges from 7 to 12 and in some seedlings the adjacent cotyledons may be joined laterally. The first order needles are not visible in sections of 34-day old seedlings. The hypocotyl is circular in outline and maintains a uniform thickness to the base of the cotyledons. The basal half of the hypocotyl is somewhat 106 K. VENKATARATNAM et al. brown and the upper half green. In transection the hypocotyl shows a uniseriate epidermis with thickened outer walls. Beneath this is the cortex composed of 5 to 7 cell layers, with intercellular spaces. The endodermis and pericycle are not distinct. The stele may be triarch or tetrarch (figure 27) with radial vascular bundles and exarch xylem. Towards the outer side on the radius of the phloem, there is a colourless mass of ceils (figures 27, 28, 29). These, according to Chauveaud (1902) and Wilcox (1962) are the precursory phloem while Schopf (1943) refers to them as the procambium. Between the phloem and the precursory phloem there is a two-layered cambium (figure 29) extending up to the pericycle and separating the xylem from the phloem and the precursory phloem. In the centre of the stele there is a colourless pith (figures 27 to 30). All the layers mentioned in the hypocotyl extend into the root and are common except that the epidermis is not true, tl-e xylem smaller, represented by 2 to 3 cells, and the precursory phloem less developed. Just above the root initials the cortical cells are arranged in regular layers around the stele. The phloem and the so-called precursory phloem c ifferentiate slightly proximal to the initials. Root-shoot transition--The vasculature in the root with 4 xylem and phloem patches undergoes the following changes upwards (figures 8 to 20). The xylem is represented by 4 patches of 2 to 3 cells each (figure 8) without any distinction into proto- and metaxylem. Phloem could not be distinguished at 2,050/~ above the root initials. At 4,050 ~ the xylem patches widen and show the exarch condition and phloem also becomes evident (figures 9, 27). At this time the precursory phloem appears, though not very distinctly, between two adjacent xylem patches and towards the outside of the phloem, the cambium being between the phloem and the precursory phloem (figure 28). Still higher up, i.e., 1,100/~ below the shoot apical initials, the xylem, phloem and the precursory phloem are well-developed This structure is maintained in the hypocotyl 825/~ below the shoot apical initials. Just below the coty-lcdonary node (figures 12, 30) the xylem and phloem widen tangentially with the precursory phloem at its zenith. Below the cotyledonary node, each xylem and phloem patch may bi- or trifurcate (figure 15) and the total number of branches is equal to the number of cotyledons. By this time the four patches of the precursory phloem unite with each other to form a cylinder around the xylem and phloem strands (figures 14, 30). At the cotyledonary node each xylem and phloem group enters the nearest cotyledon (figures 17, 18, 30) where the phloem occupies an abaxial position to the xylem and forms the primary K. Venkataratnam et al. Proc. Indian Acad. Sci., Vol. 81 B, No. 3, Plate I 1975, pp. 101-110

25 ""

Figure 22. Median L.s. of shoot apex of mature embryo 1 hr after seed wetting (x 510).

Figure 23. Median L.s. of shoot apex of 34-day old seedling (x 260). Figure 24. Median L.s. of radicular apex of mature embryo (X 165). Figure 25. Median L.s. of root apex of 34-day old seedling (x 205).

(AI, Apical initials; SAI, Subtpical initials; SL, Surface layer on the flanks; F, Flanking zone ; RM, Rib meristem ; PE, Periclinal division ; NP, Needle primordium ; S, Stele ; I, Root initials; C, Columella; RC, Root cap; X, Xylem; P, Phloem; PP, Precursory phloem; CI, Cortical initials; CA, Cambium; V, Vasculature,

( facing page 106) Anatomy of the mature embryo .... 107 vascular bundle (figures 19, 20, 31). At the cotyledonary node the precursory phloem divides into branches and each branch enters a cotyledon along with the xylem and phloem, occupying an abaxial position to the phloem (figure 31). In the cotyledon the primary vascular bundle consists of 2 to 3 xylem tracheids on the adaxial side and precursory phloem on the abaxial side with the phloem lying in between these two (figure 31). There is no direct vascular connection between the hypocotyl and the shoot apex. Hence, the root and shoot vasculature are independent and the hypocotyl is structurally like the root.

DISCUSSION Cytohistological zonation in the shoot apices of Picea smithiana, evident in the mature embryo, becomes more distinct as the seedling develops and the shoot apex enlarges. The characteristic zonation pattern of the shoot apex is clear in the 34-day old seedling. Gregory and Romberger (1972) also made similar observations in Picea abies. Though Johnson (1951) states that the apices of all investigated gymno- sperms are similar in possessing a superficial zone of initiation, a group of subapical mother cells and a flanking zone, variations occur. In the shoot apex of Picea smithiana Shah and Thulasi (1967) reported five zones, viz., the apical initials, the subapical initials, the rib meristem, the surface layer and the flanking zone. Gregory and Romberger (1972) reported a cup- shaped lightly staining apical Zone [which in the terminology of Foster (1938) and Pillai (1963 a, b, 1964) would include the apical initials and central mother cells], a peripheral zone and a rib meristem. Tepper (1963) reported a similar zonation in the shoot apex of Pinus ponderosa. Here the apical initials are dealt with separately from the surface layer on the flanks because of differences in size and staining capacity. All the surface layer cells stretching across the apical dome divide periclinally so that there is no stable surface layer as in the shoot apices of Araucaria (Pillai 1964) or on the flanks alone as in some Cupressaceae members (Pillai, A. 1963; Pillai, S. K. 1963 a). As Korody (1937) suggested, this type of apex may be considered as a "naked corpus '. The subapical initials form a small zone as compared with that in the soft pines (Sacher 1954). The peripheral zone is as usual composed of small highly chromophilic cells. The tendency towards elimination of periclines in the surface layer exhibited by some other gymnosperms (Taxodium, Cross 1939; Cupressus. spp., Pillai, A. 1963; Pillai, S. K. 1963 a; Podocarpus gracilior, Pillai, S. K, 1963 b; etc.) is absent in Picea smithiana. Evidently, P. smithiana can be grouped along with Pinus, Abies and other members. K. Venkataratnam et al. Proc. indian Acad. Sci., Vol. 81 B, No. 3, Plaie H 1975, pp. 101-110

Figure 26. T.s. of root of seedling 2,050ff above root initials (;< 205). Figure 27. T.s. of root of seedling 4,500/, above root initials (>: 160). Figure 28. T.s. of hypocotyl 825/L below shoot initials (x 205). Figure 29. T.s. of cotyledonary node (x 205). Figure 30. T.s. of portion of hypocotyl (x 360). Figure 31. T.s. of cotyledon (x 360).

(facing page 10"~) 108 K. VENKATARATNAM et aL The configuration of the radicular apex and the root apex of the seedling is basically similar. There is a common initiating region from which the columella cells arise distally and the stelar cells proximally. Surrounding this is a hollow cup-shaped group of initials. The cell files produced from this zone broaden distally by kappe divisions and abut around the columella head. The distal cells of this group differentiate into the cortex while the outer files, which proceed towards the tip of the root, form the peripheral part of the root cap. In the embryo, however, the kappe divisions of the protoderm surrounding the hypocotyl give rise to the peripheral part of the root cap. As growth proceeds, this zone is composed of ceils in continuation of the cortical files, the older files from the protoderm being sloughed off evidently. Schopf's (1943) concept of the pericolumnar cells near the stelar initials changing polarity abruptly, becoming oriented slantingly and giving rise to the cortical files proximally, cannot be supported by the analysis of the cell lineages, as has been brought out by Pillai A. (1964, 1966) in the root apices of a large number of conifers and Ephedra foliata. This cell lineage must be a continuation of the activity which started during the histogenesis of the embryo. Van Tieghem (1891) conceived the changes in the seedling vasculature as a process of rearrangement where splitting, rotation and fusion are involved. Eames and MacDaniels (1947) have described four types of transition on this basis. The xylem is described as bifurcating, splitting and rotating through 180° to become endarch while the phloem also splits simultaneously. Here the vasculature is considered as continuous from the root to the shoot. However, in Picea smithiana seedlings, there is no connection evident between the shoot vasculature and that of the root-hypocotyl-cotyledon. The vasculature as observed here is continuous from the root to the cotyledons through the hypocotyl. Bonnier (1900 a, b) thought that the xylem pole differentiation shifts gradually. In the present study, the xylem patches enlarge tangentially but the pole of xylem differentiation shows no shift. Sterckz (1900) thought that the root and shoot are fundamental morpho- logical entities which are placed in juxtaposition in the hypocotyl so that there is no longitudinal connection between the strands. The results reported here seem to justify the suggestion that the vasculature of the root and shoot are distinct morphological entities. The seedling structure of Picea smithiana with independent vasculature for the shoot and root-hypocotyl-cotyledon seems to support Thoday's (1939) interpretation of the double origin of the vascular system and not the unitary system of Eames and MacDaniels (1947). Chauveaud's (1911) ~oncept of evolutionary acropetal development i8 not supported, Anatomy of the mature embryo .... 109

ACKNOWLEDGEMENTS The authors are grateful to Prof. R.V. Singh, Himachal Pradesh University, Solan, for kindly supplying the seeds of Picea smithiana. They are grateful to Dr. (Mrs.) Ambuja Pillai and Prof. V. Purl for critical appraisal of the manuscript.

REFERENCES

Allen, G. S., Embr~ogeny and the development of the apical meristems of Pseudotsuga--II. Amer. J. Bot. 34 73-80 (1947 a). Allen, G. S., Embryogeny and the development of the apical meristems of Pseudotsuga--III. Amer. J. Bot. 34 204-211 (1947/)). AI She,rift, K. A., Histological Studies on the Shoot Apices and of Certain Cupressaceae. Ph.D. Dissertation, Univ. Calif., Berkeley (1952). *Bonnier, G., Sur l'ordre de formation des elements du cylindre central dans la racine et la tige. C.R. Aead. Sci. Paris t. 131 781 (19003). *Bonnier, G., Sur la differentiation de tissue vasculaire de le feuUe et de la tige. C.R. Acad. Sci. Paris. t. 131 1276 (1900b). Buc,hholz, J. T. and Old, E. M., The anatomy of the embryo of Cedrus in the dormant stage. Amer. J. Bet. 20 35-44 (1933). * Chauv~ud, G., De l'existence d'elements pre~urseurs des tubes cribles chez les Gymnsopermes. C.R. Aead. SoL Paris 134 1605-1606 (1902). *Chanvcaud, G., L'appareil conducteur des plantes vaseulaires et les phases principales de son evolution. Ann. Sci. Nat. IX Bet. 13 113-138 (1911). Cross, G. L., The structure and development of the apical meristem in the shoots of Taxodium disticluam. Bull. Torrey Bet. Club 66 431-452 (1939). Cross, G. L., A comparison of the shoot apices of the sequoias. Amer. J. Bet. 30 130-142 (1943). Barnes, A. J. and MacDaniels, L. H., An Introduction to Plant Anatomy. 2nd Ed., McGraw- Hill Book Co., New York (1947). Foster, A. S., Structure and growth of the shoot apex in Ginkgo biloba. Bull. Torrey Bet. Club 65 531-536 (1938). Foster, A. S., Problems of structure, growth and evolution in the shoot apex of seed . Bet. Rev. 5 454-470 (1939). Gregory, R. A. and Romberger, J. A., The shoot apical ontogeny of the Picea abies seedling--I, Anatomy, apical dome diameter and plastochron duration. Aw~r. J. Bet. 59 587-597 (1972). $ackman, V. A., The shoot apex of some New Zealand gymnosperms. Phytomorphology 10 145-157 (1960). Johamen, D. A., Plant Microtechnique. McGraw-Hill Book Co., New York (1940). Johnson, M. A., The shoot apex in gymnosperms. Phytomorphology 1 188-204 (1951). Kemp, M., Morphological and ontogenetic studies on Torreya californica---Thc vegetative apex of the mogasporangiate . Amer. J. Bet. 30 504-517 (1943). Korody, E., Studieu am Spross-Vegetationspuakt von Abies eoacolor, Picea excelsa und Pinas montana. Beitrage Biol. Pflanzen 25 23-59 (1937). Pillai, A., Structure of the shoot apex in some Cupr~sac.eae. Phyton (Redactio), Austria 10 261-271 (1963). Hllai, A., Root apical organization in $ymnosperms--Hm~ conifor~, Bull, Torrey Bot. Club 91 1-13 (1964), 110 K. VENKATARATNAM et aL

Pillai, A., Root apical organization in gymuosperms--Root apex of Ephedra foliata, with a tenta rive suggestion on the possible evolutionary trend in the root apical structures in gymno- sperms. Planta 70 26-33 (1966). Pillai, S. K., Structure and seasonal study of the shoot apex of some Cupressus species. New PhytoL 62 335-341 (1963a). Pillai, S. K., Zonal structure and seasonal variations in the shoot apex of Podocarpus gracilior. Prec. Indian Acad. Sci. 57B 58-67 (1963 b). Pillai, S. K., Structure and seasonal study of the shoot apex of two species of Araucaria. Oester. Bet. Zeits. 111 273-284 (1964). Riding, R. T., Early ontogeny of seedlings of Pinus radiata. Canad. J. Bet. 50 2381-2387 (1972). Sachet, J. A., Structure and seasonal activity of the shoot apices of Pinus larabertiana and Ptnt~s ponderosa. Amer. J. Bet. 41 749-759 (1954). Schopf, J. M., The embryology of Larix. 11l. BioL Monogr. 19 1-97 (1943). Shah, J. J. and Thu|si, K. S., Shoot apical organization of Picea smtthiana. Prec. Indian Acad. Sci. 651t 177-180 (1967). Singh, H., Seasonal variation in the shoot apex of Cephalotaxus drupacea. Phytomorphology 11 146-153 (1961). Tepper, H. B., Dimensional and zonational variation in the dormant shoot apices of Pinus ponderosa. Amer. J. Bet. 50 589-596 (1963). Tepper, H. B., Ontogeny of the shoot apex of seedlings of Pinus ponderosa. Amer. J. Bet. 51 859-865 (1964). *Sterckz, R., Recherches anatomiques sur L'embryon et les plantes clans la famille des Ranun- culacees. Mere. Soc. Roy Sci., Liege HI series, t. U (1900). Thoday, D., The interpretation of plant structure. Nature (London) 144 571-575 (1939). *Van Tieghem, P., Traite de Botanique 2rid Ed. Paris (1891). Wilcox, H. E., Growth studies of the root of incense cedar, Libocedrus deeurrens--l. The origin and development of primary tissues. Amer. J. Bet. 49 221-231 (1962).

* Original not seen.