Diversity of seed plants and their systematics
Gymnosperms II (Morphology, anatomy and life Cycle of Pinus)
Dr. Davinder Kaur Reader in Botany,
Maitreyi College University of Delhi.
Date of submission:- 10/10/2006
PINUS
Gymnosperms Coniferopsida Coniferales Pinaceae Pinus
Pinus is the most important genus of family Pinacceae which is the largest and the most recent of the modern conifer families. Pines usually grow on the slopes of hills and form dense and extensive forests of evergreen trees in the North temperate regions of the world. They are distributed mainly in Northern Europe, North and Central America, Subtropics of North Africa, the Canary Islands, Afghanistan, Pakistan, India, Myanmar and the Philipines and spread up to Indonesia. In tropical countries like India, Pinus is found in the hill with subtropical or temperate, climates, though some pines are grown as ornamentals even at lower altitudes. The species of Pinus are of great economic value and are the main Source of timber, fuel fan resins.
The genus Pinus is represented by over 100 species (Mirov, 1967). Dallimore and Jackson(1966) however mention Ca.80 so valid species. They are divided into two natural subgenera. A. Haploxylon (the soft pines) the scaly shoot at the base of short shoot is deciduous, the ray tracheids in secondary wood of stem and root have smooth wall, there is single vascular bundle in the needle (Fig 12 B) B. Diploxylon (the hard pines) the scaly shoot at the base of short shoot is persistent, the ray tracheids have corrugated walls, and the needle has two vascular boundless (Fig. 12 A,C)
In Indian Subcontinent, there are six species of Pinus of which four are distributed in the Himalayas.(Fig. 1,2) They are:
1. P. roxburghii Sar. , popularly brown as “Chir”, is the most important among Indian pines. It is very common and grows from 460 m to 1500m in the Western Himalayas, extending up to Bhutan and Eastern Nepal, either forming pure forests or in association with other taxa.
2. P. wallichiana A.B. Jacbs, the “blue” or the “Bhutan pine”, commercially known as ‘kail’, is found growing from 1500m to 3000m. it is also common in Western Himalayas in Kashmir Vally, Shimla, Chakrata and Mussoorie and in Eastern Nepal. In the eastern sector it commonly grows in the Khalaktang area, Rupa Valley and the Diarg. Dzong Valley. It is frequently associated with other conifers.
3. P. gerardiana wall, ex Lamb., the source of edible seeds “Chilgoza” or “Nioza” occur in Northern Afghanistan and North-Western Himalayas, in Tibet, Kashmir and Pakistan and Pockets of Himachal Pradesh (Kalpa and Pangi districts) at an elevation of 1830-3600m.
4. P. insularis Endl. (Syn P. Khasya), the “Khasi” pine, is restricted to the Eastern Himalayas at an attitude of 800 to 2000m in Garo, Khasi and Jaintia hills.
5. P.armandii Franch.. the ‘Armands’ pine is restricted to North eastern Himalyas at an attitude of 1200- 3600m. It grows in Arunachal Pradesh.Plants are very common in NEFA.
6. P. merkusii Jungj and de Vries, the Tenasserim pine, is the most tropical in nature out of all India pines and is found in southern Shawt states of Myanmar and hill slopes of Andaman and Nicobar islands.
MORPHOLOGY
Young pine trees area pyramidal with horizatal branches at regular intervals (Fig. 3) The tree start losing its symmetry with age and, in later stagers it may appear rounded, flat or even spreading .
Root:
Pinus exhibits two types it root system Viz the long roots having potential for indefinite growth constituting the main root system (There is a primany tap root with a large number of laterals called long roots).And the branches with restricted growth and relatively short life, called the short or dwarf roots. Some of there root branch dichotomously, have an ectotrophic mycorrhiza (Fig 6A,B). And are termed mycorrhizal roots.
Stem:
The main stem erect, woody and covered with rugged scaly bark which peels off. The branches are of the two types: A. The long shoots with unlimited growth. B. The dwarf shoots with limited growth.The long shoots occur the main stem and bear an apical bud enclosed in bud scales. Each long shoot arise as a lateral bud in the axil of a scale leaf. These lateral buds grows horizontally to a certain length and is referred to as nodal growth,. In some pines this growth produce a single internode in a year (uninodal pines) while in others (multinobal pines) two or more internodes are formed in a year. The long shoots, when fall off, leave scar on the stem.
The dwarf shoots also called short shoots, brachyblast or foliar spurs, are borne on long shoots and arise in the axil of scale leaves (Fig. 4). Each dwarf shoot bears two opposite scaly leaves, called propphyls followed by 4- 13, spirally arranged scaly cataphylls and finally depending upon the species, 1-5 needle. The needle number is constant for species and is used as a taxonomic character for identification of pines e.g. P. monophylla has one, P. sylvestris has two and P. roxburghii has three and P. wallichaina has five needle. The older parts of the long shoots are covered with scars left by the fallen dwarf shoot and the subtending scales. At regular intervals it also bears compact rings of bud scales or scars. The parting between these successive rings represents each years growth.
Leaves: The leaves are of two kinds: (i) the foliage leaves which appears only on the dwarf shoot.They are long, narrow (acicular), tough, green and known as needles. Their surface is smooth. They are borne on dwarf shoots in clustess of two to five. (In P. monoplylla only one leaf is produced on the dwarf shoot). The needles are persistent. They fall only when the spur is shed on a while (Hence the pine tree is evergreen). The needles are straight on young shoots but spread outwards or drop down in older shoots. (ii)The scale leaves; they are brown membraneous and protective in function. The scale leaves are the only ones borne on the long shoots. They are found on dwarf shoots as well. They fall off as the branches nature.
Monofoliar spar (dwarf shoot with one needle) P.monophylla Bifoliar spur (dwarf shoot with Two needles) P. merkusii
Triflliar spur (dwarf shoot with three needle) P. roxburghii, P. gerardiana , P. insularis Pentafolia supr(dwarf shoot with five needles) P. wallichiana, P. armandii
ANATOMY Shoot Apex :-
The apex of the long shoot undergoes periodic activity. During the dormant period, the apex has a low parabolic dome. When it resumes its activity, it assumes a high rate of mitotic activity and attains a tall, parabolic form. At this stage, the needles produced in the axils of the scale leaves, project out of them. The shoot apex is
distinguishable into four zones : (i) Apical initials (ii) Central zone of mother cells (iii) Peripheral tissues zone and (iv) Rib meristem (Fig. 7A). The apical initials, by anticlinal divisions contribute to the outer layer of the peripheral tissue zone, and periclinal and oblique divisions give rise to the rib miristem and the inner layers of the peripheral tissue zone. The rib meristem diplays a regular vertical aggangement of cells and later coustitutes the pith. The apex of dwarf shoot is dome- shaped and also shows four zones
.
Root:-
Long roots. In transverse section the epidermal cells appear more of less isodiametiric and many of them are filled with tannin. The broad cortex is distinguishable into peripheral zone of small and an inner zone of large parenchymatous cells (Fig.5A,B). Frequently, the cells of both the zones are filled with starch. The endodermis is composed of suberized cells, usually impregnated with tannin which gives them the brownishogange colour.
It shows indistinct casparian-strips, followed by 6-7 layers of pericyle. The walls of th peripheral pericylic cell are slightly thickened while those of the inner cells are thin. Many of them contain tannin.The stele is generally diarch or tetrarch,(Fig. 5A,B,E) by may sometimes show pentarch condition. The number of protoxylem elements varies from 8 to 16. Each protoxylem point is associated with a resin duct (Fig.5A,B,E) and consists mostly of scalariform or scalariform-pitted tracheids, while the metaxylem is made up of pitted tracheids. The phloem, which alternates with the xylem strands, consists of parenchyma, sieve cells and tannin filled cells. The pith cells contain a considerable amount of starch; some of them also contain tannin.
Secondary growth sets in when the primary tissues are still in the process of differentiation. A zone of cambium differentiates from the parenchymatous cells beneath the phloem (Konar, 1963). This by repeated periclina divisions forms secondary xylem towareds the pith and secondary phloem towards the cortex. In the region of the resin ducts, the cambium cuts off only parenchymatous cells, resulting in broad xylem rays (Fig.5B,E). With subsequent development of the secondary wood. The rays are reduced to the width of only a single cell.
The secondary xylem is made up of tracheids with bordered pits on their tangential and lateral walls (Fig.5C,D).
The rays are either uni-or multiseriate, the latter being always associated with resin ducts. The primary phloem soon gets crushed and is unrecognizable. The secondary phloem consists of radially oriented rows of cells.
Many of the parenchymatous phloem ray cells contain tannin.
As the vascular cambium is differentiated in the stelar region, a cork cambium is formed in the outer region of the pericyle. This cuts off cork cells externally and parenchyma internally (Fig.5B,E). In P. roxburghii the cork cell become highly suberized and cutinized. Several of them have their lumen filled with tannin (Fig 5B).
Dwarf roots. Structurally the dwarf root is similar to the long root except for the following differences.
S.No Long root Dwarf root
1. Root-cap present Root-cap absent 2. Ratio of the stele to cortex high Ratio of the stele
to cortex low 3. Fair amount of secondary
growth
No secondary growth
4. Starch in cortical cell present Starch in cortical cells absent
5. Resin ducts present in primary cortex
Resin ducts usually absently
Mycorrhiza : Pinus exhibits well developed ectotrophic mycorrhizal association with over 50 different species of fungi belonging to families Boletaceae and Agaricaceae of the Basidiomycets. The dwarf roots divide dichotomously and become modified into mycorrhizal system after fungal infection, when the entire roottet is enclosed by mycelium (Fig6A-C). The fungal hyphae penetrate the intercellular spaces in the cortical cells of the root forming the so called Hartig’s net (Fig. 6D). Occasionally inttracellular hyphae are also met with. As the resistance of the host cell develops, most of the intracellular hyphae are digested and the fungus remains primarily in the intercellular spaces. The pine-fungal relationship is symbiotic, the pine tree is benefited by the increase in absorbing area of the mycorrhizal root for soil nutrients.
Stem:- Young stem
A transection of a young primary stem, slightly below the apex, shows superficial ridges and furrows due to adpressing of the surrounding leaves (Fig.7B). The epidermis is followed by a broad parenchymatons cortex.
The primary, endarch, collateral, open vascular bundles are arranged in a ring and separvated form on another by broad medullary rays. The resin duets are arranged in a ring in the cortex. These arise shizogenously and have an inner living of epithelial cells. The pith is parenchymations and many cells of pith and cortex contain tannin. No. endodermis and pericyele can be distinguished.
Each bundle has a primary phloem on the outside and primary xylem on the inner side with primary cambium (Intrafascicular) in between the two. The primary xylem consists of tracheids that are arranged in uniform radial rows and xylem parenchyma only. The protoxylem elements have a loose spiral thicknening with a few small bordered pits on their radial walls. Annular thickenings are rare. The metaxylem elements are reticulate and pitted. The phloem contains sieve cells, phloem parenchyama and albuminons cells.
Secondary structures:-
The interfascicular cambium arises in the region of the medullary rays and connects with intrafasicular counter part, so as to complete the cambial ring. The cambium functions in a typically angiospemous manner and forms two types of cells. (i) The first type comprises elongated cells with tapering ends known as fusiform initials.
These give rise to the longitudinal or vertical system of xylem and phloem namely tracheids, xylem
parenchyma, sieve cells, phloem parenchyma and albuminous cells. (ii) The second type, the ray initials consist of isodiametric cells. These are much smaller than the fusiform initials and give rse to ray cells i.e. transverse system. Due to the tangential extension of the fascicular cambium the bundles also increase in width. Further activity of the cambium produce a continous zone at secondary xylem and phloem (fig. 8C). At please the cambium cuts of parnehymatous cells which form the secondary medullary rays.
Secondary exylem consists of tracheids’s with bordered pits (fig.9C-G) on radial walls and rarely on tangential walls (Fig.10) In the bordered pit, the pit cavity formed by over arching of the secondary wall is called the pit chamber and the opening in the secondary wall that faces the cell lumen is called pit aperture in bordered pit pair. The pit membrane is thickened in its central portion. This thickening which is of primary nature, is disc shaped and is termed torus. The pit membrane around the torus- the margo is porous (Fig.9G). During differentiation of the margo the cell wall matrix is dissolved so that only the cellulose mionnofibrils remain. The pit membrane is flexible and under certain conditions, the torus can be pushed against one of the pit apertures. In this condition the pit is called aspirated. Mature tracheids shows transverse thickenings between the pits, termed as crassulae or Bars of Sanio(Fig.9C). Sanio (1873) described these as rod shaped horizontal thickenings in the middle lamella. EM studies have shown them to be more refraction pattern (see Mauseth 1988).
The secondary phloem consists of sieve cells (sieve elements) which arise directly by transformation at the phloem initials. Rarely the pholem initials divides once to give rise to tow sieve cells. The sieve cells are arranged in rows. The most characteristic feature of the sieve cell is the occurrence of sieve areas on the radial walls (Fig.8D,E). Through out the length of the sieve cell. Some times two or more sieve areas many be gwaped together. The siever areas consist of numerom narrow channels (0.1-0.5 um in dia) lined with callose. The sieve elements are in close contact with Albuminom cells through a rather specialized pore.
The vascular Rays:-
The vascular rays are initiated by the ray initials of the cambium and once formed, they continue to increase in radial length indefinitely. The detailed structure of array can be understood by examining sections cut in three different planes (TS, TLS, & RLS) (Fig.8B;9A-C;10). The rays are two type: (i) uniseriate rays, and (ii) multiseriate rays. (Fig.9B) Most of the rays are unisenate type and varies from 1 to 12 cells in height. The multisenate ray is always more than one cell wide and several cells in height. It is associated with a centrally started resin duct. The medullary are hetrocellular composed of two types of cells. (Fig.9C). The starch filled central cells, termed ray parenchyma, are living. The peripheral cell are, thick-walled, dead and called marginal ray tracheid or tracheidal cells. These cells have no protoplasmic contents and are further distinguished from the ray parenchyma in having bordered pits instead of simple pits. In the phloem region, the ray consists of the usual centrals cells, which are living and store starch, and marginal cells which are also living but store only albuminous matter.
The Transeverse section (TS) shows the width and the length of the rays. The tangential section (TLS) shows the height and width of the ray and radial longitudinal section (RLS) length and height of the ray.
Every year the activity of the cambium attains its peak in the spring. The xylem elements produced during this season are fairly broad, but with the onset of summer, the activity is slowed down and the diameter of the elements becomes appreciably smaller. This results in the formation of growth rings (Fig.8A;9A;11) commonly called annual rings (counting the number of these rings given an indication of the age of the tree or branch). The thick walled, narrow, squrarish, compact summer wood of one ring passes rather abruptly in to the thin-walled broad and some what polygonal tracheids of the following spring.
Resin ducts are distributed all along the wood: maximum occurrence of the ducts is seen just before the onset it summer (Fig.9A;11) these are all lined with prominent epithelial cells (Fig.8C) sometimes a resin duct may become blocked by the enlargement of the epithelial cells, such structure and termed tylosiods .
Periderm: -
As the vascular cambium is being differentiating in the stellar region, a cork cambium arises in the first or second layer of the cortex. It divides periclinally and gives rise to the cark or bark on the periphery, and the secondary certex towards the centre. By intermittent auticlinal divisions it increases in circumference of the stem. The scaly bark thickens gradually, cracks externally and ultimately wears away.
Compression Wood: It is formed along the under surface of branches, on th trunks of learning trees. It apparently forms in response to geotropic stimuli and may also be due to the concentration of auxin, as shown in P. strobus by Wershing and Bailey (1942)
Dwarf Shoot:- Anatomically, the dwarf shoot resembles a long shoot except for its narrow diameter. It has a small cortex, few resin ducts, and the vascular bundler are open and collateral. The medullarly ray
broad and parenchymatous. The tracheids are mostly scalariform, occasionally pitted. A large parenchyma tons pith has many cells filled with tannin.
Leaf :
The plant bears five types of leaves in sucession:
(i) Cotyledon, (ii) Juvenile, (iii) Prophyll (iv) Catphyll (v) Acicular or needle
The cotyledonary leaves are the first to arise, their number varying from 4-15. In transverse section it is almost triangular with the sides generally longer than the base. Juvenile leaves follow the cotyledonery leaves and assume the normal function of leaves for 1-3 years. Each leaf has a broad quadrangular outline. Prophylls and cataphylls appear sickle shaped with broad marginal wings. Anatomically both prophylls and cataphylls show the same character. Acicular or needle libe leaves are borne on the dwarf shoot or spur after the development of prophylls and catapylls.
The needle shows a complex internal structure. In a cross-section it is shaped like the tri-sector of circle with the curved surface facing outwards and the vertex inwards (Fig.12AC). A single layered epidermis, forms, the outer boundary. Its cells are thick walled, lignified and is covered on the outside by thick cuticle. The deeply sunken stomata are present on all sides of needle i.e. amphisotomatic. Below the epidermis, one or more layers of sclerencymmatous hypodermis is present which is frequently interrupted by airspaces beneath the stomata.
Mesophyll tissue lies in between hypodermis and endodermis and shows no differentiation into palisade and spongy tissue. Its cell are peculiar in the their walls have numerous small infolding which project into the cavities of the cell. These cells are thin walled and contain numerous chloroplasts. The mesophyll contains a number of resin duct immediately under the hypodermis (Fig.12B,C). The central portion of the needle is occupied by stele which is enclosed by a conspicuous single layered endodermis which delimits the measophyll.
Its cell are large, oval with thickenings on the outer walls. The pericycle follows the endodermis. The number of vascular bundles in needle may be one (P. sylvestris, P. wallichiana ) two or more (P. roxburghll, P.contorata).
Generally the hard pines have two vascular bundles in their needle while the soft pines have only one when two bundles are present, they are disposed at an angle and are separated by band of sclerenehyma tissue. Each vascular bundle contains, the phloem faces the curved or outer side and the exylem lies facing inwards i.e.
towards pointed end. The vascular bundles is collateral. The protoxylem is partly crushed in the mature leaf. The metaxylem is composed of helically thickened and bordered pitted tracheids. The xylem elements are in radial rows and are interspersed with rows of parenechymatous cells. The phloem pasrnchyma is more abundant than that of xylem. Many of its cells contain starch, others have protein and few may also have crystals.
On either side of vascular bundles are present special kind of cells which constitute the transfusion tissue (Fig.12C). Embedded in the parenchymatous pericycle are richly cytoplasmic cell which abut upon phloem, are called albuminous cell. The other type cells are radially elongated and resemble the tracheids. These are knows as tracheidal cells. (the transfusion tissue consists of parenchymatous, albuminous and tracheidal cells). And are interspersed with parenchyma and albumious cells.
Xerophyitc characters of leaves:
The plant generally grow on slopes where the rain water easily drains off. The low temperature of winter also interferes with efficient root absorption causing a physiological draught.The leaves therefore, exhibit typical characters of xerophytes i.e.
(1) The transpiring surface is reduced (2) The stomata area sunken
(3) Cuticle is very well developed
(4) Epidermal cells thick walled (Lignified walls) (5) The hypodermis is sclerenchymatons and (6) The vascular system well developed.
The Reproductive Cycle
Strobili
The plants are monoecious. The male and female cones are borne on different branches of the same tree(Fig.13A,B). Male cones occur laterally in clusters, each in the axil of a scale leaf at the base of a terminal
vegetative bud (Fig.13A;16A-B).The male cones replace the dwarf shoots at the base of the developing bud or shoot of the current year and are spirally arranged on long shoot. Each male cone is shortly stalked and consist of an elongated central axis, bearing a number of small spirally arranged and closely fitting scale like microsponophlls with their scaly apices upturned (Fig.16F). The microsporophyll is attached to the axis by a short stalk and bears two microsporangia on its lower (abaxial) side (Fig.16E,F). The microsporangium dehisces by a longitudinal slit. (The young male cones of P. roxburghii are about 1.5 cm-2 cm long and 1 cm in diameter. At the time of dehiscence these are 2.5cm – 3cm in length.)
The male cones arise on the lower and the female cones on the upper branches. The female cones replace the long shoots (Fig.13B) and are generally borne in pairs, but the number may go up to six (for female cones see Figure 14A-E; 15A,B).
Each female cone consists of a central axis which bears paird scales 180-90 in number) in a close spiral (Fig.15C, F-I ) The lower scale of the pair is small known as bract scale. It is leathery and directly attached to the cone axis. The upper scale of the pair is larger, thicker and stouter. It is the ovuliferous scale/
megasporophyll. The bract and the ovuliterous scales together form a seed-scale-complex (Fig.15G,H). Each ovuliferous scale bears two-two ovules on the upper or adaxial surface (Fig.15D). The ovules are inverted with the micropyle facing the cone axis. The bract & scale is larger than the ovuliferous scale before pollination, but in post pollination stages the ovuliferous scale overgrows the bract scale (Fig.15 I). The ovuliferous is woody and wedge shaped with its broader sterile end, the apophysis directed outwards. The bract scales are thus concealed from view. The surface of the cone is marked by rhomboidal areas each with a small central conical point- the umbo (Fig.14E). Sporophylls at the apex and base at a cone are generally sterile. It takes approximately twenty-seven months from the time the female cones are initiated to the shedding of seeds. Seeds generally have conspicuous wings (Fig15J) excepting a few like P. gerardiana where the wings are rudimentary.
Microsporangium.
A transection of young microsporangium shows a mass of meristematic tissue surrounded by an epidermis. One, two or more cells of the hypodermal region, by repeated divisions, give rise to an archesporial tissue which has dense cytoplasm and prominent nuclei (Fig.17A). The peripheral cells of the archesporium undergo periclinal divisions and cut off the primary parietal layer towards the periphery and sporogenous cells on the inside.
Further periclinal divisions in the parietal layer forms an outer three- or four-layered wall and the inner mass of sporogenous tissue (Fig.17B). The innermost layer of the wall cells develop into the tapetum (Fig.17C).
Simultaneously, the epidermal cells undergo anticlinal divisions; most of the cells are filled with tannin except two rows of smaller cells, which form the line of dehiscence (Fig.17C). At the time of dehiscence of the microsporangium, the epidermis attains its maximal development with fibrous thickenings, and functions as the exothecium (Fig.17E) As the microspore mother cells initiate divisions, the epidermal cells lose their nuclei, become filled with a homogeneously staining substances. The outer wall of the epidermal cells gradually becomes cutinized.
The tapetum is of the secretory type. Young tapetal cells are richly cytoplasmic and multinucleate (Fig.17C).
They Iecome very conspicuous during meiosis in the microspore mother cells, and degenerate soon after the spores are released from the tetrads (17D). The tapetum is involved in the nourishing of the sporogenous cells/young microspores, and in the formation of exine on the spores.
The development of tapetum and sporogenous cells takes place simultaneously. There appears to be a correlation between the stage of the microspore mother cell (meiocyte) and the structure of tapetal cytoplasm.
Accordingly, three phases of development of tapetal cell can be distinguished corresponding to pre-meiotic, meiotic and post-meiotic phases of microsporogenesis. (For details see Dickinson 1970, 1971; Dickinson and Bell 1972, 1976; Willemse 1971).
Microsporogenesis:
Prior to meiosis the cytoplasm of microspore mother cells and tapetal cells looks alike. (Dickinson & Bell, 1976). However, with the anset of meiotic division, the mother cell wall lyses completely. At diplotene stage, the callose starts enveloping mother cells. The deposition of callose is brought about by the activity of the Golgi bodies which give out a large number of vesicles at deplotene stage. Callose, being a substance of low permeability, isoslutcs the mother cell from surrounding influences, thus helping the micropore mother cells to follow an independent course of development during meiosis. The nucleus of the mother cell undergoes reduction division, which is followed by simultaneous wall formation to give rise to a tetrad of microspores (Fig.18A-E). At the end of the division, the callose sheath is dissolved by enzymatic action and the young haploid microspores are set free (Fig.17D;18E).
Microspore: Each microspore has large nucleus and soon after the formation of microspores, a special wall is secreted around each of the spores which results in the formation of on outer exine (cappa) and inner intire (capulla). Later, the exine expands at two points on opposite sides of the microspore. This results in a cavity formed between the exine and intire which later forms saccii (wings or air sacs) in a mature pollen grain. The saccus becomes beautifully marked by the formation of delicate, irregular ridges over the entire surface expect along point of fusion (Fig.18E-I).
The pollen grains are heteropolar and rediosymmetric and consists at a body or corpes and the air sacs or sacci.
It has a single aperture or tenuitas at its distal end. The wings or sacci are separated from the body or the corpus by the saccate nexine. The nucleus lies towards the distal end (Fig.18I).
Male Gametophyte :-
The uninucleate microspore is the first cell of male gametophyte. The microspore nucleus cuts off a small lens- shaped prothallial cell towards the proximal end and a large central on the distal end (Fig.18F) These are initially separated by an ephemeral callose wall which is later replaced by a cellulosic wall. The central cell cuts off a second prothallial cell and an anthendial initial (Fig.18G) Both the prothallial cells are ephemeral. The antheridial initial now divides to give rise to a small antheridia cell and a large tube cell. (Fig.18H) The pollen grains are shed at the four celled stage (two degenerated prothallial cells an antheridial cells and tube cell).
The development of the pollen grain wall has been studied in Pinus sylvestris (Willemse, 1971) and P.
banksiana (Dickinson, 1971) by using EM. The exine consists of an outer sexine and inner nexine. The nexine, in turn, consist of nexine I (on outer side, i.e. towards sexine) and nexine II (on inner sides i.e. towards intire.
The outermost, layer, the sexine and nexine I are laid down when microspores are still at the tetrad stage and within the callose wall. A space is formed between plasmalemma and callose layer by the contraction of former.
This space is much enlarged in the region where, subsequently air sacs would develop the sexine is first laid over the area where sacci are destined to be formed; it then slowly spread over the whole area, except in the region of the germinal pore (Fig.18I). Long stretches at distinctly trilamellar tapes at lamellae are seen at this stage at development at primexine. The next layer to be laid down is nexine I. It is formed by the deposition of sporopollenin on trilaminar tapes, in the inner side of primexine. Lipid granules are the primary source of nexive I. The nexine surrounds the cytoplasm except at the region where wings would develop. Marten & Waterkeyn (1962) studied the wing formation and found them to be the extension of outer exine only. After the formation of nexine I, the microspores come out of the callose wall which is enzymatically degraded. The sporopollenin continues to be deposited on the sexine layer. Furhter development of the pollen wall takes place when the microspores are liberated from tetrads. Small granular particles at sporopollenin present between the plasma lemma and nexine I coalesce to form nexine II. Nexine is thick on the germinal pore side. Intine is the last layer to be laid down. It consist of two layers viz., an outer and the inner intine. The inner inter is a continuous layer as compared to the outer entire which is discontinuous. The inner intine which is next to the cytoplasm, is thicker at the germinal pore (distal end) and thinner at the region of prothallial cells (proximal cells). It is made of cellulose and pectin. The incomplete outer entire covers the inner entire except at the germinal pore. The inner intine is responsible for the formation of pollen tube.
The development of air sac has been traced in great detail in Pinus banksiana by Dichinson & Bell (1970). Like exine their formation starts when microspores are still with the tetrads and primexine is beinglaid down between the plasmamembrane and the callose layer.
Megasporangium / ovule
First Period of Growth of the Ovule:
The ovule is unitegmic and crassinucellate. The integument is free from the nucellus except at the chalazal end, and forms a symmetrical micropylar tube well beyond the level of the nucellus (Fig.15J). AdaxialIy, the edges of the integument extend into two long arms, which curve inwards before pollination, outwards during and curve back after pollination and finally dry up. There is no vascular supply to the ovule (Konar and Ramchandani 1958).
Megasporogenesis. The archesporial cell becomes distinguishable while the female cone is still covered by the scale leaves (Fig.15A). It differentiates at the broad apical end of the nucellus and divides transversely to give rise to primary parietal and primary sporogenous cell. The former undergoes both vertical and transverse divisions, so that the sporogenous cell is pushed deep into the nucellus, and later functions as the megaspore mother cell (Fig.19A). Starch grains accumulate at the chalazal end of the megaspore mother cell (P. sylvestris).
The latter undergoes meiosis I and produces a dyad (Fig.19B). Only a triad (Fig.) is formed if the upper dyad cell does not undergo meiosis II, or a tetrad if both the dyads undergo meiosis II. In a triad and linear tetrad, the
upper dyad cell and the adjoining megaspores or the upper three megaspores in a tetrad usually degenerate. The chalazal megaspore functions (Fig.19C) so that the development of the gametophyte is monosporic.
Three to five layers of cells round the functional megaspore become densely cytoplasmic with prominent nuclei.
This is the spongy or nutritive tissue. The spongy tissue comprises a definite zone of physiologically active cells which are concerned in the nutrition of the young gametophyte, especially at the resting stage. The cells of this zone contain abundant starch.
Female Gametophyte (First Period of Growth). The functional megaspore enlarges and shows a large vacuole even before the nuclear divisions commence. It forms only a few free nuclei (Fig.19D) (maximum at 32 nuclei) and apparently remains inactive for 8-9 months (first period of rest).
Pollination.
Each tree produces an enormous number of pollen grains dispersed by wind. The surrounding area becomes clouded by the yellow powder (also known as sulphur shower). However, only a few pollen grains reach the pollen chamber and develop further.
A pollination drop is secreted at the flared-out tip of the integument. The secretion starts a few days after the female cones emerge out of the scale leaves, and the ovuliferous scales separate sufficiently to permit the free entry of pollen (Fig.15B). Under high humidity and cell turgor, the secretion begins around midnight, and by early morning the micropyle dries up. The arrival of the wind-borne pollen at the micropyle is purely a chance phenomenon. The pollen is caught in the pollination drop, grains stick to the two-pronged micropylar canal and migrate to the nucellar tip(Fig.21.1).
The secretion of the drop is reported to be a cyclic (24 hr cycle) phenomenon. The secretion normally continues for a few days or till the time of pollination. In the presence of pollen there is a permanent withdrawal of the exudates. The cells at the apex of the nucellus are involved in the secretion of the drop. Mc William (1958) made a chemical analysis of the pollination drop of Pinus nigra and reported that the fluid contains three sugars namely D-glucose, D-fructose and sucrose.
Doyle and O'Leary (1935) and Doyle (1945) have studied the mechanism of pollination. According to Doyle, the wings orient the grain on the hanging pollination drop in such a way that the germinal pore/ furrow of the grain faces the surface of the drop. This orientation is particularly necessary since the ovules are inverted. The pollen then floats upward (due to buoyancy caused by the air sacs) and reaches the nucellus with the germinal furrow faceing the nucellus. The pollen tube enters the nucellus without curving or twisting. McWilliam (1958) emphasizes that in P. ellioltii, P. nigra and P. wallichiana, the stickiness of the neck and arm of the micropyle may be due to either a local secretion, or sugar residue resulting from the retreating micropylar fluid. This is an effective method for retaining the pollen at the site, and the prime mover of the grain is the active absorption of the fluid. McWilliams did not observe any preferred orientation of pollen on the nucellus.
At the time of pollination, the integument is four-to-five-layered, and soon after the pollen grains reach the nucellus, the micropyle closes due to a rapid division and enlargement of the cells of the inner layer of the integument.
Second period of growth of the ovule:
The female gametophyte resumes its activity in the following year in the month of February (P roxburghii) or a little later in the trees at higher altitudes (P wallichiana and P gerardiana). The additional tissues formed in the ovuliferous scales after the resumptionof growth are green. Thus both the bract scale and ovuliferous scales become very much enlarged and the umbo of the oveulifenous scale are pushed up.
Female Gametophyte (Second period of Growth)
As the ovule increased in size, the gametophyte also enlarges. The free nuclear divisions in the latter continue (Fig.19E,F) until the gametophyte occupies the entire basal and central part of the nucellus. During early stages, the nuclear divisions are synchronous. The number of nuclei in a free nuclear gametophyte is constant for a particular species; in P roxburghii and P wallchiana it is about 2500 and approx. 2000 in P gerardiana. With the enlargement of megaspore, a vacuole develops in the centre gradually pushing the cytoplasm along with the nuclei towards the periphery. At the end of last mitosis, every nucleus gets connected to six neighbouring nuclei by the formation of secondary spindles. Wall formation taken place through alveoli (Fig.19G,H ). The newly synthesized walls are unevenly thick and grow centripelally as open tubes or pipes from megaspore cell wall till they reach the centre of the vacude. Each alveolus has a nucleus at its mouth and directs its growth. Now the cross walls are laid down on each alveolus forming uninucleate cells till the entire gametophyte becomes cellular.
Development of the Archegonium:
A few cells (2-4) at the micropylar end of the female gametophyte become large and prominent by accumulating protein and RNA, and function as archegonial initials. (Fig.20A) Each archegonial initial soon divided into a large central cell and a small primary neck initial (Fig.20B). The latter divides by two vertical walls at right angles to each other to form a neck of four cells. The neck cells are arranged in a single tier (P wallichiana, P roxburghii) or a transverse division in them results in eight cells in two tiers of four cells (P austriaca, P Montana, P rigida). The central cell envlarges very rapidly so that numerous valuoles are formed. This is referred to as the “foam stage” of the archegonial development (Fig.20C). The central cell nucleus, meanwhile, divides into an ephemeral ventral canal cell and a large egg cell (Fig.20D-E). A gradual accumulation of archegonial cytoplasm takes place with the disappearance of most of its vacude. The developing egg cytoplasm contains a number of densely staining, protein rich bodies (Fig.20D-F) which hare been variously termed as proteid vacuoles, paranuclei and Hofmeister’s granules. Electron microscopic studies (Camefort, 1962) have shown that traditionally known proteid vacuoles are basically membrane bound cyloplasmic bodies of two sizes:
(a) small inclusion, and (b) large inclusions (Fig.20G). The small inclusions are portions of general cytoplasm, partly enclosed by a single membrane. These inclusions maintain connections with the general cytoplasm by short peduncles. The large inclusions are less frequent, bound by a double membrane. The included cytoplasm contains dictyosomes, mitochondria and small inclusions. Camefort (1966, 1968) reported that the large inclusions are portions of cytoplasm surrounded by highly deformed and hybertrophied plastids. Thus, in the egg of Pinus one can distinguish two types of cytoplasm: (a) “fundamental cytoplasm or the general cytoplasm, and (b) enclosed cytoplasm. The newly formed egg nucleus is Feulgen – positive, but as it increases in size, it loses its Feulgen. positivity so much so that a mature egg is completely Feulgen-negative. EM studies on developing egg have shown an interesting relationship between the egg nucleus, the mitochondria and formation of perinuclear zone. (for details refer: Camefort, 1962, 1967, Chesnoy and Thomas, 1969, Thomas & Chenoy 1969, Moitra 1980).
The gametophytic cells near the apical end of the individual archegonium grow vigonously resulting in the sinking of the latter. Thus each archegonium has its archegonial chamber. Cells of the gametophyte surrounding the archegonium form a special covering layer, the jacket. Numerous pits are present on the inner thickened walls i.e. facing the archeogonium. The oarchegonium maintains contact with the surrounding tissue through these pits.
Megaspore wall: It is prominently developed. The exine of megaspore wall consists of an outer sculplured sexine and an inner smooth nexine. The sexine is piloid and the pila are free from each other.
Post-Pollination Development of Male Gametophyte.
The Pollen deposited on the nucellar apex germinates immediately.The pollen tube arises from the inner pectin-celluosic layer at intine or inner intine (Martens & Waterkeyn, 1962) While moving through the nucellus the pollen tube secretes enzymes such as pectinase and cellulose dissolving the nucellar cell walls (Willemse & Linshen, 1969) As the tube emerges, the tape nucleus is the first to move into it.
Renewed activity of the male gametophyte in the second year:
Initially, the growth of the pollen tube is very slow as compared to the rapid development of the ovule. The apicul part of the nucellus, which had been penetrated by pollen tubes during the previous year, does not become active again. The cells at this part become thickened and empty.And the cells lose their contents. The underlying cells are meristematic and rich in starch. Due to their growth, the tip of the nucellus with the pollen tubes is lifted above and away from the developing female garnetophyte.
Towards the end of March or later, depending on the species (P. Maheshwari and Konar 1971), the pollen tube resumes active growth. In P. sylvestris the nucellar cells adjoining the pollen tube degenerate; the hemicellulose and pectin are affected first (Willemse 1968, Willemse and Linskens 1969). During this period, the antheridial cell in the pollen grain divides to form the stalk and body (spermatogenous) cell (Fig.21.2D,E;21.3A). However, there is much variation in the time of division, not only in different species but also in the same species.
Willemse and Linskens (1969) observed that, following the division of antheridial cell (called generative cell by them), the stalk cell has fewer organelles as compared to the body (spermatogenous) cell. Also, the cytoplasm of the stalk cell is vacuolate, but that of the body cell is dense. The stalk and body cell migrate into the pollen tube.
Soon after, the body cell divides into two male gametes. To begin with, both the male gametes are equal, but later, one of them enlarges considerably (Fig21.2E,F;21.3C). These two unequal male gametes have a distinct cytoplasmic sheath around them. The male gametes are Feulgen-negative, stain faintly for RNA.
Fertilization
The pollen tube, just before fertilization, grows rapidly and reaches the neck of the archegonium (Fig.22A,B).
The tip has a dense cytoplasm, a large number of starch grains, tube nucleus, stalk cell and the two male gametes enveloped in a common cytoplasmic sheath (Fig.22A,B). The pollen tube penetrates into the neck of the archegonium, and discharges its contents at the tip of the cytoplasm of the female gamete (Fig.22C).
One of the two male gametes moves towards the female gamete. The second male gamete, along with the stalk cell and tube nucleus, and the original male cytoplasmic sheath, persist at the apex of the egg and eventually degenerate (Fig.22D). The functional male gamete is devoid of the cytoplasmic sheath as it moves down the archegonium to fuse with the female gamete (Willemse and Linskens 1969). The mitochondria and plastids brought by the pollen tube are morphologically different from those of the female gamete. They remain grouped in the upper part of the egg cytoplasm during the division of the zygote (Camefort 1965, 1967). The functional male gamete lodges in a depression on the female gamete (Fig.22D). As soon as contact is established, junctions between the nuclear membranes of the two gametic nuclei are established at several points. The two nuclei establish contact by minute pores which develop in the nuclear membranes (Chesnoy and Thomas, 1971) These bridge of communication gradually increase in size and number. The zygote initially is separated from the general cytoplasm by the nuclear membrane. By the time first mitosis tabes place, the nuclear membrane disappears. The two free nuclei come to lie within the neocytoplasm (neo = new/which is jointly contributed by the nucleoplasms at the male and female nucles, and is entirely different from the mother (archegonial) cytoplasm. The embryo develops in this neocytoplasm.
Embryogeny
Proembryo development:
The zygote nucleus divides mitotically to give rise to two nuclei. The division is intranuclear and the resulting nuclei are formed within the nucleoplasm of the zygote (Fig.23A,B). The next division immediately follows resulting in four nuclei which move to the base of the archegonium (Fig.23C,D). A third synchronous division results in eight free nuclei. Secondary spindles develop and wall formation takes place and give rise to an upper group of four cells, the primary upper tier (pU) and a lower group of four cells, the primary embryonal tied (pE).
The pU tier has no wall towards the upper side and is, thus open (Fig.23E,G). Internal divisions in both tiers results in four tiers of four cells each (Fig.23F,H,I). The lower two tiers comprise the embryonal tier (E) followed by disfunctional suspensor (ds) previously termed the rosette tier. The uppermost tier, U, is the upper derivative of pU. This is open above and was earlier referred to as the open tier, a term which is no longer used.
Embryo differentiation:
The upper four cells of the E-tier elongate and function as the embryonal suspensor (Es), and the lower four cells form the embryonal mass (Fig.24A;25A-E). Subsequently, there are several layers of Es, which are termed Es1, Es2, Es3 …., formed by divisions in the E-tier. The Es cells elongate rapidly and thrust the terminal cells (embbryonal cells) forward into the corrosion cavity. The latter enlarges and facilitates elongation of the Es.
Later, as the elongation of Es exceeds the rate of enlargement of the corrosion cavity, the Es becomes coiled and twisted (Fig.25F-H). The elongation of the Es system pushes the growing embryonal mass into central portion of the female gametophyte. As development proceeds further, the earlier suspensor system collapses, while newer Es are formed.
Polyembryony is common in Pinus. Additional embryos originate by simple polyembryony, i.e. from multiple zygotes, as well as due to cleavage polyembryony, i.e. a single zygote gives rise to multiple embryos by cleavage or splitting of the embryonal tier E. The separation into embryonal units occurs at Es2 formation (see H. Singh 11978) These units remain unchanged during the earlier stages of Es elongation, but later they divided and form a multicellular mass. There is a period of embryonal selection due to competition between the four embryos from each zygote, and between the embryos from different zygotes. This phase of competition lasts for about 6 weeks from the time of fertilization (P. Maheshwari and Konar 1971). Generally, the deep-seated terminal embryo succeeds and develops further. The remaining embryos become arrested at different stages of development.
The embryonal cells of the developing embryo divide in various planes and formed embryonal mass which is a smooth paraboloid structure. It has a hemispherical apex at its distal end (Fig.24B,C;25D,E), and a suspensor which is continuous with it at the proximal end. Further development at the proximal end gives rise to a well- developed root cap. It differentiates independently into a column and a peripheral region; the former does not contribute to the development of the latter.
As the root cap and root initials organize at the basal end (Fig.24D,E). An incipient pith is recognizable in the hypocotyl between the epicotyl and the root initials. It is evident by the predominant transverse divisions, cell enlargement and vacuolization. The cortex becomes demarcated soon after. The formation of pro-cambium is associated with the early phases of cotyledon formation though it is well developed before distinct cotyledonary primordial originate. Simultaneously, several long, uninucleate and multinucleolar secretory cells become distinguishable in the cortical region. Finally, 3-18 cotyledons and the shoot apex differentiate (Fig.25H,I). The cotyledons are traversed by the procambial strands, and show development of mesophyll. Generally, only one embryo matures, occasionally even two may reach maturity. The mature embryo has distinct epicotyl; root axis with remnants of the suspensors, and a hypocotyl-shoot axis bearing several cotyledons. (Fig.26)
Structure of the Seed:
There is a outer hard shell or seed coat (testa) which protects the inner parts of the seed (Fig.26). In a young ovule the integument comprises three layers. As the ovule develops further, the integumentary cells divide and become differentiated into three zones: (i) The outer fleshy zone of 7 or 8 layers of cells with shrunken nuclei and vacuolated cytoplasm its outermust 2 or 3 layers get filled with some shiny granular material, (ii) the middle stony zone of 18-20 layers of cells with lignified walls, and (iii) the inner fleshy zone consisting at 7 or 8 layers at thin and elongated cells.
The seed is winged and the development of the wing is closely linked with the development of the seed coat.
There are however some species which possess rudimentary wings (P flexilis, P. cembra) or are wingless (P koraiensis, P cembroides). The outer layer of the integument and part of the ovuliferous scale contribute to the formation of wing. The wings are normally thin and papery, but in some species they are thickened at the base.
The wings may be adherent or easily detachable.
Within the seed coat is the brown papery tegmen (derived from nucellus) The latter surrounds a white fleshy female gametophyte (Analogous to endosperm) which surrounds the embryo.
Towards the pointed end of the female gametophyte is the nucellar cap which represents the remains of he nucellus. During development both embryo and female gametophyte store reserve food materials which are med at the time of germination.(Please refer Hakansson 1956, Simola 1974, Mia 2 Durzan 1974 Durzan and Chalupa 1968, Kalsuta 1961, Hatano 1957, Kanar 1958, Nyman 1966). In the centre of the endosperm is a distinct central cavity in which lies he embryo. The embryo consist at a short axis differentiated into radicle towards the micropylar end. The hypocotyl forming a major portion below the cotyledons and the stem apex the epicotyl (plumule) which is surrounded by 8-13 cotyledons. The tip of the radide is attached to the dried up suspensor.
Dispersal and Germination of Seeds:
The pine seeds are normally dispersed by wind to long distances. Water sometimes may help in dispersal along the streams. Cones may also roll down higher elevations thus establishing plants at lower elevation.
In such of the pine species where the female cones remain closed at maturity, the seeds remain viable for a long time. Seeds of haploxylon pines lose their viability quicher than those of diploxylon pines. When stored at low temperature around 50C, seeds beep viable for a longer period then bept at room temperature. It has been suggested that changes occurring in the unsaturated fatty acids in pine seeds appear to be connected with the loss in their viability (Mirov, 1967).
The germination at seed is epigeal (Fig.27). The food stored in the seed breaps down during germination and is the only source of energy for the germinating embryo. Several changes to be place after the seed has imbibed water. (for details please refer Durzan et al 1971, Simula 1974, Ching 1970, Lopez-Perez et al 1974, Konar and Moitra, 1980).
Temporal Considerations (see Fig. 28A,B)
In P. roxburghii (growing at an altitude between 500 and 2500 m in the NW Himalayas, India) the male cones are initiated in September. Pollination takes place (pollen grains at the four-celled stage) in March. Pollen germinates on the nucellus immediately after shedding. There is a period of rest for approximately 10 months (from May to February – 2nd year). Growth is resumed in March (2nd year) and fertilization occurs in April (2nd year). The female cones are initiated in February. Soon after pollination in March, the cone undergoes a period of rest from April to January for ca. 10 months. The female gametophyte is at the free-nuclear stage at this time.
Growth is resumed from February (2nd year), and fertilization occurs in April. The embryo developes and matures by December (2nd year). The cones open and shed their seeds in Apirl-May (3rd year). Usually, the cones dehisce in the third year; the undehisced cones may also contain mature seeds.
There are three species of Pinus – P. pinea, P. leiophylla and P. torreyana – which have a 3-year-type reproductive cycle. These taxa undergo three winter rests, and the interval between pollination and fertilization is 2 years. The cones are initiated during autumn and the ovules overwinter (1st rest period). Pollination occurs during spring when the ovules show sporogenous cells. Megasporogenesis occurs during autumn and the ovule with a slightly enlarged megaspore overwinters (2nd rest period). The megaspore matures through the coming spring and summer. It undergoes free-nuclear divisions during autumn. A massive spongy tissue develops around the megaspore, and the young female gametophyte. The ovule overwinters again (3rd rest period) at the free-nuclear gametophytic stage. In the coming spring, the gametophyte develops more rapidly and fertilization takes place by June. The interval between pollination and fertilization is 2 years. The embryogeny is completed and the mature seeds are shed in autumn or early winter.
The reproductive cycle in tropical and temperate pines is conditioned by the environmental factors and, therefore, the 2 and 3- year cycle.
Suggested Readings:
1. Bhatnagar S.P.& Moitra A. 1996 Gymnosperms. New age International Ltd. New Delhi.
2. Bierhorst, D.W. 1971. Morphology of Vascular Plants. The Macmillan Co., New York.
3. Biswas, C. & Johri B.M. 1997. The Gymnosperms. Narosa Publishing House. New Delhi.
4. Chamberlain, C.J. 1935. Gymnosperms: Structure and Evolution. Chicago Univ. Press, Chicago.
5. Dallimore, w.& Jackson, A.B. 1966.A Handbook of Coniferae and Ginkgoaceae (revised by Harrison, S.G.). Edward Arnold Ltd. London.
6. Gifford, E.M. & Foster, A. S. 1989. Morphology and Evolution of Vascular Plants. 3rd edn. W.M.&
Freeman & co., New York.
7. Khan, R. 1940. A note on “double fertilization” in Ephedra foliata. Curr. Sci., 9:323-324.
8. Khan, R. 1943. Contributions to the morphology of Ephedra foliata Boiss. II. Fertilization and embryogency. Proc. nat. Acad. Sci. India, 13:357-375.
9. Konar, R.N. 1960. The morphology and embryology of Pinus roxburghii Sarg. With a comparison with Pinus wallichiana Jack. Phytomorphology, 10:305-319.
10. Konar, R.N. 1962. some observations on the life history of Pinus gerardana Wall. Phytomorphology, 12:196-201.
11. Maheshwari, K. 1960. Morphology and Embryology of cycas. M.Sc. Thesis, Univ. Delhi.
12. Maheshwari, P. & Konar, R.N. 1971. Pinus (Bot. Monogr. No.7). Council Sci. Industr. Res., New Delhi.
13. Moitra, A. & Bhatnagar, S.P. 1982b. Cytochmical studies on the reproductive structure of Indian gymnosperms. III. Microsporogenesis in Pinus roxburghii. J. Palynol., 18:33-42.
14. Plant, D.D.1973. Cycas and the Cycadales. Central Book Depot., Allahabad.
15. Plant, D.D.& Das, K. 1990. occurrence of non-coralloid aerial roots in Cycas. Mem.
16. Singh, H. 1978. Embryology of Gymnosperms. Engylopaedia of Plant Antmy. Vol.X. Gebruder Borntraeger. Berlin, Stuttgart.
17. Singh, H. & Maheshwari, K. 1962. A contribution of the embryology of Ephedra gerardiana Wall.
Phytmorphology, 12:373-393.
18. willemse, M.Th.M. 1971a. Morphological and quantitative changes in the population of cell organelles during microsporogenesis of Punus sylvestris L. I. Morphological changes from zygotene until promeatphase I. Acta Bot. Neerl.,20:411-427.
