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A HISTOGENETIC STUDY
OF THE
NEEDLES OF PINUS PALUSTRIS MILLER
By
ROBERT OSBORNE THOMAS
A TESS PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF. FLORIDA
IN PARTIAL FULFILMNT OF THE REQUIREMENTS FOR THE
DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
September, 1942
ACKNOiu rWLDlGNT8
The writer wishes to express his gratitude to
Professor i.D. Gody for his guidance during this inves-
tigation. e also wishes to presss his appreciation to
Professor W.B. DeVall for additional advioe ooneerning the
arrangement of materials; to Drs. E.B. Sherman, H.S. Rolfe
and A.M. Laeasle for the use of their camera equipment and
to iMr. Randolph Wedding for his assistance aith the pho-
tography; and also to those who have contributed helpful
suggestions pertaining to the histological teohuique.
Botany Department
University of Florida
august, 1948.
TABLE OF CONTENTS
Page
I. INTRODUCTION........................ 1
II. SIURVY O Tim LITIRATU. ......... ....... 4
III. JAThRIALS AND MTHODOS..................... .... 6
Selection of material for study.............. 6
Colleotions...... ............................. 7
Growth measurements..........*..*.......** ......
Preparation for aiorosoopio study.............. 10
Killing and fixing........................... 10
Dehydration and olearing....................* 18
Paraffin infiltration........................ 15
ikmbeadding.................................... 16
Seotioning..*****.......,............ .**... 16
Fixing the sections to the slides.,.......... 17
Staining techniques employed................ 17
Methods of recording results.................. 18
IV. RESULTJ AND DISCUSSION......................... 19
The bud and the various foliar appendages...... 19
External development......................*... 24
General description of bud development
and needle emergene.*............*........ 24
results of growth measurements............... 89
General anatomy of the needle............ ..*.* 84
Histogenesis of the needle..............* .... 42
Page
Characters of the aploal merittema.....,,.... 42
Initiation and early development
of the needle priaordiua,.................. 46
Origin of the cortioal tissue................ 47
Inorease in thickness and initiation of
prooambial tissue.,,,. ,,,0,............... 49
Interoalary elongation of the needle.,,,,,., 51
Differentiation and development as seen
in serial transeotions................... 53
Further features of development.............. 70
Comparison with angiospearm.................. *74
SU ARY.......................................... 76
VI. BIBLIOGRAPHY........................*** .* ...*..** 78
VII. BIOGRAPHY
TABLb OF CO~UTATS (oont'd)
ILLUSTRATIONS
Page
Fig. 1. Fasciole of young needles to show
method of laying off segments.............. 9
Fig. 2. Suotion apparatus employed during
fixation of pine material................ 14
Fig. 8. proliferating apur shoots of F. Palustris..... 21
Fig. 4. Longitudinal section of bud showing
component parts...............**........* 822
Fig. 5. kn'largeient of spur shoot including
scales and primordia........................ 23
Plate I. Characters of bud and bud scale
in P. palustris...................... 25
Plate II. Stages in development of terminal
leader bud...*....................... 26
Plate III. Stages in development of lateral
branch bud........................... 37
Fig. 6. Stages in needle emergence and relative
position of sale and emerging fasoiole..... 28
Plate IT. Needle growth by segments.............. 31
Graph 1. Length of needle segments at ap-
proximately weekly intervals......... 32
Table 1. Measurements on needles of P. Ralustris
showing elongation of segments...... 35
Fig. T. Needles of P. palustris illustrating
marginal serrations and orientation
of stomata......*....**..........*.........* 36
Plate V. Anatomioal characters of mature needle.. 38
Fig. 8. Jear median longitudinal section of
apioal aeristem of spur shoot............... 43
fig's. 9-12. stages in early development of
primordiunm........................... 45
ILLUSTRATIONS (oont'd)
Page
Fig. 13.
Transeotion of primordium 300 microns in
length taken midway between base and
apex.......................................
Plate VI. Diagram of near median longitudinal
section of young needle fasoiole
3000 microns in length with four
enlargements of various portions.....
Plate VII.
Transections of young needles re-
presenting the first three stages
to traoe tissue differentiation.....
Plate VIII. Transeations representing stages
4, 5 and 6 in the same series......
late XX. Transeotions representing stages
7, 8 and 9 in the same series.....*..
Plate i. Transeotions representing stages
10, 11 and 12 in the same series.....
Fig. 14.
Transeotion through basal portion of
needle 320 millimeters in length..........
Fig's. 15-20. Diagram of near median longitudinal
section of needle 3000 microns in
length including apex of spur shoot
at base, with enlargements of various
portions to illustrate further
features of development...............
72
INTRODUCTION
The pine needle is extensively employed in both elemen-
tary and advanced botanical studies to exemplify one type
of extreme xerophytic adaptation, so that definite infor-
mation regarding its growth and differentiation should be
desirable. modern botanical texts and periodical litera-
ture, however, reveal that histogenetio studies of the
leaves of Pinus have been somewhat neglected. Aiost of
the literature concerning histogenetio studies of foliar
organs deals with various genera of the angiosperms. The
majority of papers on the leaves of gymnospermous plants
deal with mature leaf structure and concern anatomical and
taxonomic characteristics, but exclude histogenetic features.
The values usually attached to the information brought
forth in various histogenetic studies of leaves indicate
a wide range of possible usefulness. Knowledge of this type
has in different papers been considered of importance to
other phases of botany, such as genetics, pathology, physi-
ology and evolutionary history of the higher plants. Yet
the writer is of the opinion that the structure of the
mature pine needle is so extreme in its departure from
that of the typical leaf that it warrants a histogenetio
study for the value of the information itself, aside from
the possibilities of its application in other fields.
Although this problem has been investigated from a purely
academic point of view, there may exist other values of
the material if it can be used to gain a better understand-
ing of the attacks of certain rusts on needles of our
southern pines, or if it contributes to the knowledge of
the evolutionary history of foliar development among the
more primitive seed plants.
It has long been known that the foliage leaves of
angiosperms and gymnosperms in general develop from a
meristematic tissue out off from the growing point. These
leaf primordia have the power to develop the same tissues,
and yet the story of their differentiation in different
species indicates a wide range of possibilities inherent
in them.
Since the needle or foliage leaf of Pnus palustris,
or for that matter of most of the pines, presents an ex-
treme in the characters exhibited by its mature tissues,
it is but natural to wonder what peculiarities in its develop-
ment are responsible for the final structure which varies
so greatly from that of the typical leaf. The following
problems, which arise in the histogenetio study of any leaf,
are of special interest in the case of the pines because
of the abnormal condition in their mature leaf structure:
(1) In its developmental history, when do the
various structures become apparent, and what differences in
development underlie variations in the mature structure?
(8) Oan the divergences in shape of the cells
forming the mature tissues be correlated in any way
(a) with the time and also the relative
amount of development of the various tissues,
(b) with the differences in rate of nuclear
division in the various leaf tissues,
(o) with the type of metabolism of the cells
of the different tissues and
(d) with external conditions?
These are some of the problems of leaf development
whioh seemed to be outstanding, and the study was planned
to interpret the developmental story of the leaf selected
with such problems in view.
In carrying out the study it was proposed to (1) select
a representative species for the investigation; (2) oolleot
its buds in various growth stages up to and including the
"candle" stage, then oolleot the fascicles of young needles
in various stages of development from the time they start
elongating within the fasoiole sheath up to and including
needle maturity; (5) prepare microscopic sections at various
levels and planes through the buds and needles of varying
ages to show histological origin and development; and (4)
record results by means of drawings, and photomicrographs
where desirable.
The main objective nas been to discover what peculiar-
ities in development are the causal factors in producing
the final structure of the mature pine needle which varies
so radically from that of the typical leaf.
It is well to review some of the literature pertaining
to this subject, although little of it is directly concerned
with tais phase of study on the lower forms of seed-bearing
plants.
SURVEY OF THE LITERATURE
The increasing number of papers dealing with histo-
genetic studies of foliar organs indicates a recent stimula-
tion of interest in this method of study. Most of them are
concerned with the angiosperms. Several studies have been
made in order to secure data of use in the interpretation
of certain periolinal chimeras (e.g., Buder, 1928; Krumbhols,
1925; Lange, 1927; Noaok, 1922). Later studies by Cross
(1936-1938), Foster (1955-1937), Kliem (1957), R8sler (1928),
Zim2erman (1928) and others have added to our knowledge of
the development and basic nature of the leaf and the bud
scale. Newman (1936) and Grigoirc (1958) have applied this
method to studies of the floral parts of several flowering
plants. most of this literature is comparatively recent
and has been thoroughly reviewed by Foster (1936), who has
emphasized that few detailed studies of foliar organs along
comparative lines have been made in the past.
Although considerable study of the leaves of gymnosperms
has been made, earlier investigators have, for the most part,
pXaoed emphasis Upeoa aatO ieal sa tazonoie oharsetors.
I7 Thomas (8S) was the first amoag these earl woukesr
to *all ahoation to tho easato8iel haraeters of th
leaves of eetonifts u a mass o olasslieatleai. Other
authors on this sUbj*t have bees DWrtU~ (18Q), iMfb
(1SMS), aeolbmsam (1MO), a1 Ooseitu r ua Blas (MeW).
ngelomaI is tohe mot familiar of these Ua seaetiea with
thi groa of d gaImspe reate us w f the hbaruo ase
obtabie ftrl leaf str eture to tdei e y a of ahis sb
tiviaio of Lthe gams gJn e. Oseulte ad Ioe later
TverIlA his reomult sat made aUs of thoee elseters tIn
theM Udlelatateofr sp eoes. Del We Newkuae (ISM)
ae poes~it tho mat dilig t lavestliaters smum lter
marlws *e have ued eLhtf~etes f o lft a mateep tbh
i detif e-tie at piMe. Thy MaST a et hL talue letN
., o
aOf the salte eatree whtih ise of 4aneste yes
map (4"m) has ms a slalu statB ef t *nhe *
pusoe.aM has pebtas -t musWsses&bsa
$mlm .t40 b-*. Va on e
be et 'L'"abe 3 .
miftiir *** ^.*-* ^'v**N~c*9^ I.
^^Mi -.alM "^''^'I^^ ^IIJIBIJI
__4 1 r Allilll
0001 1P
-LIM
r"TWW r l r W
..T All
a 'a r
rk i
r. 4
V fltS4
a. A
iL ts. .'~H I')j7.
r.~
:u-~-- 4F~ rrlII
r~r )'I I m low
*!O ito7 .14,1 ia- .,, p"
ge Mell 4
V
M A
. a.. :w: ee e 4p~
-lrw .LgiEb r # -Lus @1 t,'~ i UY~lmu~q~ *
.. llt,. -. .21-21 U1~ -t ~ 'ldr~~t.qY~~
C.lrtvl'' .*.Il~sP~r~' V."--
*L~wL~lr~lWr~ 'qaC~i~)~I 1~~~~tl~
.~~prr~ u il ~r
-C -y 4 Idl~,r~r kL r~ II
Ia~ r
-.y ~ V :
I; *'r ~ ~ ,*.
AlC
*aft 4t~seftsimas the "ba of t wtest elongation or the
jloaer inLeas Nw tho wie hm as fessiou"L sheath.
4m; mes vwt Sol"", NAVOS wamesm this besw .Me
won LaJwOd woowr on at"to5 w. =to to Wn. tkO
ObMW 1110 tsioS ug motw 4 1tblhe se$ as orans.
tw UMotLa tho oet "ea of 61"O3USt. sOu'd to
USo bSftiSa so mw .w of oovweh shestS"M
~. 4t9!n4~ov.In lomph Whns they ms Iwjfte.A
4*81 Mir t wo th mu at34 af WahA
4g the- beit l b qiua m Vitus "a ou*at
~St~s~-a ftuwei l~o ofGI Othe Somem low t"0 ~
-ItM S 4
a r~-w
~ i~~%l%~l~qi")''~D)I~~iL r~ Idrr ~ ,w
.~l~-;~lrP~ Ir I ~r
1 etn 8 .ae a ualt, tor alated by the mark whieh originally
haL 4aivi4d the a eoles in half. 8 emnts 3 and 4 were out-
side ef the sheath at the beginnalg and oould be measured
aepeaately. Roferenoo may be made to figure 1 for a better
aema ptisa of the'iethwo of laying ottf these segments.
IIll ll llll IIII IIllll ll llljll llljl ll Illl ll lll |l
LAsORATORY' n
APPARAtUS 3O,
saw wro J ut 3086" o ars .
.J m iilflmlll iil l iiflml llJI1J1 liI
i
X .* (mastswmL sas) a tea
at Ml tmsr t
'* Al meauremets wer sado with a veroner ealiper, th
seownts were martkd and their original measureonts reoorded
on April 89, 1941 subsequent easurements wore made at weekly
intervals throughout this growing season until October Oth.
It was thought best to wait until the young needloa had
emrged a diatanoo equal to the length of the ftasiilar
sheath before they wer divided into seG nts, so that four
portions of approxaately the sam lengths 4ight be 14d off,
two of shih would be inside and the other two outside of the
sheath. mseAe, the firt part of the growth marve for thoee
neod3e is laskrng*. te results presented ar averages of
measuroments mado of needles looatod oa the mid-portion of
the shoots of several young treS growing on the oenpa
fhis data lnd iated only the approxiaato loeaties of the
region et needle eloagation.
IauIam 1~Z n 11 xo00iorZo aSOt..
heo solootion of a suitable reagent for killing
ad facing the tissues ot the maure pine needle prosenteo
no ait altioSm es of t tho standard fixatives ealfoyed It
boatnaea iwo eoteebhnieq we a satsfoaty. bioweve, the
Uame is tho bud paseaatea difforoet problem e to the
peat N eollqerml osr ss P"60 *ell wa thieoAess -d the"
of the 4 pt3eplast amB btew e i fe bVudss pet*1
s *; m*** f"miis se a as of ps ,
,. p.
S ** ..,f *,
greatly beouoo of the oharaoter and arrangement of their
sealoes these fit very closely together in the early stages
and later spread apart to permit the lodging of air bubbles
in many mall oarities. This inoreases the difficulty of
penetration.
Various killing and fixing reagents have been reooon
mended for meristematio tissues. Ball (1941), in an Iln
tenoive study of iaoroteobnioal methods of the shoot apiece
of palms, was confronted by the same problem neountered
here, namely, to seoure thorough penetration to all parts
without the ooeurrenoo of pronounced undulations in the
*ell walls and without shrinkage of the protoplasts. He
experimented with numerous killing and fixing agents of
various oonoentrations and finally recommended for best
results either the intermediate strength of formal-aeetio-
aleohol (f.A.A.), as prescribed by Ohamberlain (1932, 1965),
or the intermediate strength of formal-ohrom-aoetic solution
(7.O.A.). He also reooomanded the use of reduced pressure
for the removal of air bubbles so as to secure a more rapid
and thorough penetration.
Preliminary tests wre made by the writer with portions
f the terminal buds of lm taood. llinag and fixing
agents containing formalin, ethyl alcohol and glacial aeotie
sacd were aploed in varying proportions and ooneontrations.
Ohum-ae*tie solution (10) and Oaray's fluid, prepared a
described by Ohnauerlain (195M, 1955), were employed aels
for comparison with the former series as to thoroughness
of penetration and the amount of plasmolysis, as well as
o11 wall undulations produced in the meristematio tissues.
Oarnoy's fluid gave the most rapid penetration and
faoilitated the infiltration of further reagents by dis-
solving the oopious supply of resin usually present just
following the handle stage. However, this reagent was
not employed on further material because it produced marked
plasmolysis in the oells of the growing point and foliar
primordia.
No striking difference was observed between material
fixed in F.A.A. solutions and that in 1 per sent ohrom-aoetie
solution exoept that penetration was apparently ore rapid
in the former reagent; this was probably due to the pres-
ones in the F.A.A. solution of alcohol, which lowered the
asurfas tension, The shroaaeSetio solution has a water
base and thus the resin, together with the air bubbles it-
prisooed behind the bud sales, prevented this fluid frem
readily eateriag the butd s at. Although keeping the
material uder reduced pressure by mans of a filter pump
greatly alleviated the diffttisty this latter reagent was
considered inferior to intermediate FA.A.. solution for
the particular material studied. This oonolusion is eon-
rfned by the wide experience of Johansen (1940) in fixing
a great variety of gymospera tissues.
All material subsequent to that employed in the pre-
lininary tests was killed and fixed in F.A.A. solution for
84 hours. The following combination of intermediate strength
gave beet results:
6 oe. formalin
89 oo. ethyl alcohol (70O)
5 so. glacial aoetio acid
Aspiration with a filter pump proved advantageous (see figure
2) in all eases when removing air preliminary to the fixation
of pine needle and bud material, The amount of air contained
in both of these organs is surprising, Its immediate removal
facilitates complete penetration of the fixative into all
tissues..
2. Dohyhation and clearing.-
Some experimentation was nade with the purpose of
arriving at a method for dehydrating this particular material
which would embody the following advantages: not be so
drastic in its action as to cause plaamolysis or cell wall
undulations; not harden the tissues and thus make them more
refractory when being sectioned; and possibly simplify the
oomonly drawn-out procedure using the ethyl alcohol and
zylol series (Ohamberlain, 1932).
A dehydration series reooeaonded by Zirkle (1950) was
tested. It involves the use of ethyl alcohol and n-butyl
alcohol in increasing oonoontrations for both dehydration
and clearing. The results indicated no apparent advantages
over the conventional aloohol-zylol schedule. Pine buds
treated with Zirklles dehydration series remained as re-
fraetory as when treated in the ordinary manner, and the
procedure was neither shortened nor simplified to any great
extent.
Figure a. Apparatus for sabjeoetag material to
reduced pressure while in killing and fixing
fluid and also in dehydrating agent. filter
pup attaohed to faueet; J. stop-cook valve; g.
water trap; container with reagent and material
being treated.
The schedule adopted employed cellosolve (ethylene
glycol mono-ethyl ether) as the dehydrating agent (Hanoe,
1959) and normal butyl alcohol as the clearin- urgent. Ax-
oessive hardening of the tissues was prevented by the ad-
dition of 5 per cent glycerine to the cellosolve. Three
changes of cellosolve at intervals of 6 hours each were
made. As soon as the materials were fixed they were washed
in running water for 6-8 hours, after which they were in-
troduced directly into cellosolve; then they were subjected
to suction with an aspirator for 15-20 minutes. following
the third change of oellosolve the materials were cleared
by playing them in n-butyl alcohol for 12 hours, and then
changing to fresh butyl alcohol for an additional 12 hours.
Paraffin infiltration was then started.
8. Paraffin infiltration.-
Zirkle's (1930) method of paraffin infiltration was
employed. The material was transferred to a vial filled
two-thirds ftill with paraffin. It was then covered with
butyl alcohol and kept in an oven (40-42 deg. C.) over-
night. Next the butyl alcohol-paraffin mixture was replaced
with melted, filtered paraffin (m.p. 56-56 deg. 0.). Four
hours later it was replaced with fresh paraffin of the same
grade. During these time intervals the vials were kept
in an oven adjusted to maintain a temperature of 56 deg. C.
The material was embedded as soon as no trace of the butyl
alcohol could be detected.
4. Imbedding.-
Imbedding was carried out with freshly filtered
paraffin. The technique of Brough (1959) was employed be-
cause it proved valuable in preventing crystal formation
in the wax. The imbedded material was cooled and forced
as quickly as possible beneath the surface of the ice-water
to harden. Then the paraffin block was removed froa the
tray and stored in the ice-cube compartment of an electric
refrigerator for one or two days in order to set the paraf-
fin properly.
5. Sectioning.-
Portions of imbedded pine buds and needles were
refractory if sootioned immediately after hardening of the
paraffin. This was due probably to the hardening effect
of dehydration, clearing and paraffin infiltration upon
the tissues. The sections of such material were often
torn or irregular in thickness, but when treated according
to the technique of Cutuly and Cutuly (1954) this diffi-
oulty was removed. This technique consisted of triimmin
the paraffin block so that a portion of the imbedded tissues
was exposed, after which it *as pluoed in a vial of dis-
tilled water, stoppered securely, and allowed to soak for
three to fourteen days. The length of this period depended
upon the nature of the material and also on the size of the
pieces which were imbedded. The water softened the imbedded
tissues and thereby facilitated the sectioning. Although
the co-authors of this method worked with animal tissues
the procedure w's found to Aork equally well with pl2nt
materials. Johansen (1940) recommends it for many plant
materials ahich are relatively soft and easily cut at the
tiaue of collecting, but become rather tough and difficult
to section when carried through the different stares of
preparation for imbeddinf.
The sections for this study were cut on a Snenrer rotary
microtome. Most of the rateriae wia secticned at 8-10 mi-
crons. Unless otherwise specified, the illustrations are
of sections cut at this thickness.
6. Fixing the sections to the slides.-
The adhesive qualities of the fixatives usea were
compared. deotions of buds fastened to the slides with Mayer's
egg albumin preparation (Chemberlain, 1932) proved unsatis-
factory in many instances because they loosened during the
staining operations. Consequently Haupt's gelatin fixative
(Johansen, 1940) wes employed as a substitute and found
superior to the albumin preparation. Bud scales, which
before hud loosened most readily, held securely upon the
slides throughout the staining schedule.
7. Staining techniques enployed.-
The different stains used for these sections vere
safranin in separate combinations with Delafleld's haema-
toxylin, mention violet, Bismarck brown (Chamberlain, 1932),
and fast green TCP (Moore, 1936). Among the special stains
employed were Boke's (1939) nodificati:n of the D:l-;.i Id's
haematoxylin-safranin schedule, cross' (1937) Aodiflc-Atlon
of safranin 9nd fast Preen, Foster's (1974) technique 'or
meristematic tissues usin- tnnnic icid and iron r:1loride,
and also Johansen's quadruple (19l9) and quinturle (1941)
combinations, Elthough certain nodificctions ;irre nuc in
the case of Johansen's procedures. These chen7oe *re iade
in both of his scheduled and consistrd of:
(1) iethyl violet 2B replaced by cryt-,l violet.
(2) Methyl cellosolve replaced by ethyl cellosolve.
(3) Tert. butyl alcohol rezlacEU, by n-butyl alcohol.
Johansen's stain combinations proved the most exacting
for differentiation of the mature tissue in later stages
of needle development. Best resul'i .ecre obtained on
meristematic tissues in the bud and :oun; needles with
Cross' schedule. almost equally well stained preparations
resulted with Boke's technique. However, more satisfactory
photomicro ;raphs were obtained fro.r sections stained by the
f,,rmer schedule.
MTHODS FOR HEOORDING RESULTS.-
All photomicrographs were made with a Spencer microscope
and a Leitz vertical photographio attachment. Critical il-
lumination was obtained with a Bausch and Lomb ribbon fila-
ment lamp. photography of sections stained with Boke's
hasmatoxylin and safranin series produced sharply aooen-
tuated cell walls with panohromatic film and a light green
filter. 'est result. on riifrni-n nA '"ast -rcen sitan'-
tissue? 'ere obtained with 'rntten :.' 1l~*.es mnd a yratten
" filter, no. 25. Line drrv'in .:re -Ad ":it'l '"c: ;
of an Addinger drawin- azr..t s.
hbULTr AiND DISCUSSION
THL BUD AU;D THi VARIOUS FOLIU APPU:iaGAuS.-
The order in whloh foliar ap endge.- devclo,), beginning
with the embryonic -plant, is as follows: dhen the pine seed
germinates the cotyledons develo? on the young shoot -.nd are
followed by development of .-insle juvenile leaves whioh be-
come progressively smaller ai tie shoot elon~ates, until ul-
tiaately they are mere scale-leaves. It is in the axil of
these scale tv.t the dsur shoot or dwarf branch arises.
after r the latter ha.i developed a few scal~- and : tuft of
needles it generallyy hecoren dormant. If, however, the ex-
tremity of the main axis is injured the spur shoots na" grow
into normal branches producin- at first isol-ted juvenile
green leaves and then spele leaves with spur shoots in their
axils. This injury to the btex of the main axis aay cause
the noraully dormant s1ur shoots immediately beneath to pro-
liferate, re .ulting in the production of a new shoot between
the needles of each fascicle, as is shown in figure 5.
This proliferation of the spur shoot in pines has been
observed by Dickson (1885) on P. sylvestris, in which case
injury was caused by the removal of the extremity of the
tranoh. Dortlwlick (1899) t-.te:; t' t v.riou:z inj:ri .nM
cause this :henomenon, niicn, which are prunin-, J--foli~tion
by Insects, and 1:te frosts. Dufrenoy (1918) also observed
this rejuvenescence of the spur shoot in the case of F.
maritime. Thompson (1914) states that this proliferation
is observed mo.;t frequently in seedling and youn- vigorous
plants, and su'.c is borne out by the present wr''er's ob-
serv> tions. Various pstaies of proliferated spur sLoots
have been observed in this vicinity upon seedll:-i; of _.
taeda, in which saw-flies had been the otuse of t:e injury,
and u-oni young trees of atlustris 1.ill., Iln 'icch case
the injuries. affection thi~ ..ro. t: had bee, caused by t.e
writer's removal of the ter:.inal bud immediately above
dr!ngS his collections earlier in .'he season. The results
of this letter case are shown in fir-ure 3.
Although this -ro .tAi riay not be uncommon, it is at
least abnormal. Ordinarily tle s;ur shoot remains dormant
after it Las produced a feu scales and a fascicle of needles.
The pine bud itself los-:esses a structure which h dif-
fers fro., that of the other conifer.. inoe a clear con-
ception of foliar histo enesis for any ;ivei species should
include as a background so,-e idea of the general structure
of the vegetative bud and its component parts such will be
considered briefly.
2he ve6etAtive bud of pines is in reality a compound
bud. It is composed of a main cauline axis covered with
*COe
Cm
A
sirAlly arranged, closely: i: ric:ted bud scales in t--
axils of -lhich the spur shooter are produced, .nd t'( Lutter
-ire extensions of the nain ".xis. Sao. spur shoot fir:t
develops bud :c.l.:z which envelop the newly foriaed needles.
:ence the spur shoot, together lith its various foliar ap-
pendag*s,, is in ronlit: a bud itself. Consequently the
leader as a whole is a oo:rnound bud hearing: soals nW spur
shoot buds in the axils of these cmles. The relAti')nship
between these various ippenda'es is illustrated in fl-ures
4 and 5.
6
Figure 4. Near median longitudinal section of
bud showing the closely imbrioate nature of the
bud scales and the spur shoot or secondary buds
which arise in their axils. 120.
spirally arranged, closely: imbricated bud scales in the:
axils of which the spur shoots are Produced, and the latter
are extensions of the main axis. Each spur shoot fir:t
develops bud scales which envelop the newly formed needles.
Henoe the spur shoot, together with its various foliar ap-
pendagss, is in reality a bud itself. Consequently the
leader as a whole is a compound bud -earing sales and spur
shoot buds in the axils of these scales. The relationship
between these various appendases is illustrated in figures
4 and 5.
Figure 4. Nemr ndisa rLeituainal section of
bud showing the closely labrioate nature of the
bud sales and the spur shoot or sooondary buds
whioh arise in their axils. 20O.
~~p:s
k-igure 5.8muho usicuOigabl n
o f thre s Su ro ~ s h o o t n n l d i e ao a z ; a n
p fim r il ? W ~ ~ ~ mW t l u .o t r r a a a c
EXTiRNAL DJVEWLOPMNT.-
1. general description of bud development and needle
DeVall (1941) has aptly described the external features
of the buds of this species as follows:
"Pinus palustris produces buds at the end
of ear h branch wL.ioh are covered with brown
scales. It is a common characteristic of
the species to develop several lateral buds at
the base of the larger apical bud, especially
on the terminal leader of the stem......The
base of each bud scale sl light brown and this
color darkens conspicuously at the attenuated
tip. The margins of the seale have long,
silvery cilia which impart a 'oob-webby' ap-
pearance. The oilia become entangled as the
scales elongate and become reflexed."
The production of lateral buds at the base of the larger
terminal bud is quite characteristic of this species as
compared with the other closely related pines. This feature
is shown in Plate I, figure 1. An enlargement of a portion
of a terminal bud is shown in Plate I, figure 2 to illus-
trate the arrangement of the bud scales and the appearance
of the fascicles of needles in their azils. Special features
of the bud scale, suoh as the reflexed and attenuated tip,
are shown in figure 5 of Plate I.
The buds of the subsequentt season's growth appear in
early summer before the needles of the current season have
ceased elongating. The buds develop during the summer and
fall, and not until late winter does the so-called handlingg'
of the shoot occur. The term 'candle" as applied here refers
to a bud which has reached nearly maximuu elongation but which
Plate I* Characters of bud and bud scale in P. pa1utrine
Figure 1. Terminal bud with lateral buds
at its base. 1 2/3.
Figure 2. Portion of terminal bud enlarged. X 2.
Figure 3. Dorsal and lateral views of bud scale
with accompanying spur shoot bud. X 5.
.1
Plate II. Stages in development of terminal leader bud.
x i.
-2
I~I
Plate III. Stages in development of lateral branch bud.
1 *.
III.
as yet shows no fascicles of needles. plates II and III
show various stages in the elongation of the bud and the
emergence of the fasoicles of needles. The candle is much
longer in the case of buds on terminal leaders (Plate II)
than those on lateral branches (Plate III).
Following the candle stage the needle fascicles begin
to emerge in the axils of the bud scales, thus forcing them
apart. Figure 6 shows stages of the eaerlence of the
needle from the fascicle sheath.
I
(
Figure 6. Stages in needle emergence. j. Very
short bud containing at its tip needles almost
ready to emerge from their soaley sheath; f.& 1.
fascicles separated from the bud, f showing
dorsal view of scale at base, and then 3 showing
lateral view of this same stage; 4. stage showing
needles nearly half-way out from The enveloping
sheath.
Externally the growth exhibited by the needles is prin-
oipally that of elongation or a linear foray of growth.
They emerge from the fasciole sheath as they push out be-
tween the spirally arranged scales. The final covering of
the needles is composed of the innermost scales. They are
quite thin at first and extend during the growth of the
needles until they are transparent, so that the green
needle tips can be seen even prior to their emergence.
The needles toward the base of a leader emer-e first
and maintain an early lead over the needles located along
the mid-portion of the leader. The last needles to emerge
are those located in the apioal region. Thus, there is
a wave of growth which progresses fro' the base of the
leader to the tip.
2. Results of ronth measureaents.-
It is ooamon knowledge tiat, after an initial period
of rapid elongation, the needles of conifers proceed to grow
from an active zone at the base (dohUepp, 1926; Lorody, 1957;
BUsgen, 1931). This fact is borne out by a record of growth
measurements obtained by the writer to supplement the histo-
genetio data. These measurements were first taken when the
young needles approximated 4 centimeters in length, of which
fully half was still within the fasciole sheath at the base.
Segments were marked off on these needles as has already
been described. It was found that segments 1 and 2, which
were measured as a unit, contained the area in which elongation
occurred, while there was no later increase over the initial
length of segments 3 and 4. Evidently all elongation had
oeased in the apioal half of the needles at the time of the
initial measurements. The changes in the relative length
of the segments are illustrated in Plate IV and are also
presented as a graph. A complete growth ourve for these
needles has not been given because no data were obtained
before the needles were 4 centimeters long.
The purpose of the data given is to indicate the general
region of elongation of the needles. This region falls in
the basal portion whioh is within the fasciole sheath and
is thus protected from dessioation and meohanioal injury.
Plate IV. Needle growth by segments. IX /5.
From left to right and top to bottom are represented
successive stages of needle elongation by segments
as played off and measured in procuring growth data.
Sl
tt
I
Zr
.
&; ,
Oraph 1. length of needle seamnts at apmr-oi-
mately weekly Intervals*
Table 1.
Measurements (in centimeters) on needles of
P. palustria showing elongation of segments.
Segment No.
Date 1 2 I 4
Length Length Growth** Length Length
-, .9 1 .0 __ 97 1 i7
-
fl- __
* All suooeeding length measurements
represent ooabined measurements on
8.
in this column
segments 1 and
** Growth figures in this oolump represent differences
between oonseoutive length measureomnts.
GIOENAL ANATOMY OF THE kI LMNDL.-
The anatomy of the pine needle is well known since
it is included in many elementary and advanced botanical
studies. Variation of needle micro-characters has been
studied in detail and has been used as an important means
of distinguishing the many species of this genus.
Coulter and Rose (1886) described these characters
briefly and separated the needle structure into three regions:
the cortical, the mesophyll and the fibrovascular. Included
in the oortical region were the epidermis, stomata and the
"hypoderma". The mesophyll region consisted in their de-
scription of the highly infolded chlorenohyma and the resin
ducts imbedded within it. The fibrovascular region was de-
scribed as either one or two vascular bundles surrounded by
a very distinct bundle sheath. No mention was made of an
endodermis, as such, although the bundle sheath was said to
consist of cells ".....which are either comparatively thin-
walled, or with the outer wall excessively tnickened". This
latter in reality describes the conspicuous chain of cells
which more recently has been termed the "endodermis", for
in some species its cells are uniformly thin-walled while
in others the outer wall is decidedly thickened.
This summary by Coulter and Rose is descriptive in a
general way of the structure of the pine needle. Later
workers, among whom Harlow (1931) may be cited, designate
the tissues embracing the epidermal and hypodermal layers
as the dermal region. Harlow also designates the inner
vascular tissues as the stellarr region", and states that
the endodermis with its variation among different species
may be of some diagnostic significance. He describes the
individual bundles as consisting ".....of xylem and phloem,
the latter usually appearing.........as a dark patch ex-
terior to the xylem".
The foreZoing review gives a general idea of the
tissues of any pine needle as viewed in transaction. The
mature needle of P. palustris Mill. possesses sia cro-charao-
ters which are fairly typical of those found in the hard
pines, and its anatomy will be considered here. The general
outline of leaf transeotions of a 2-needled fasoiole is semi-
oircular while that of 3-needled fasoioles is broadly deltoid.
The adazial surfaces lying next to the other needle or needles
of the same fasoiole are ih each case flat, while the ab-
axial surface lying to the outside of the fascicle forms an
are. The aggregate of needles which compose a fasoiole
always forms a complete circle. T'he epidermis consists of
a single layer of small, pentagonal cells. Usually the cell
cavity is all but obliterated by the extremely thickened
walls; however, there is occasionally one which has not
undergone this excessive thickening. The epidermal cells
are highly outioularized on each surface of the needle.
86
Figure 7. Needles of Ziul plustria illustrating
marginal serrations and distribution of stomata.
The hypodermis includes one or two layers biformm) of cells
immediately beneath the epidermis. In this species the
outerr.st layers of hypodermal cells characteristically
fail to undergo the excessive thickening exhibited by those
deeper within (Plate V, figure 1). The epidermis and hypo-
dermis form the dermal region which by its protective nature
serves to check dessioction.
At intervals the cereal layers are interrupted by sunken
stomata. They occur in longitudinal rows on each surface
of the needle (figure 7), and the guard cells are oriented
so that tneir longitudinal axis is parallel with the main
axis of the needle (Plate V, figure 5). It will be observed
that the deeply sunken guard cells (in Plate V, figure 1)
appear in transaction and are supported b, the larger sub-
sidiary cells above them. The stoma is characterized by
an inner and an outer vestibule. The outer vestibule is
formed by the subsidiary cells supporting the Tuard cells,
while the inner one, or substomatal cavity, is a space beneath
the guard cells which in many cases is bordered by the two
segments of an unfolded mesophyll cell. This feature is
shown in figure 1 of Plate V. The guard cells have a cal-
lose thickening, while the subsidiary cells possess a
thickening of cellulose on the wall bordering the outer
vestibule. These various characteristics of the dermal
region are exemplary of the pine needle's extreme adapta-
tion to xerio conditions.
Plate V. Anatomical characters of mature needle.
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Transection midway between base and apex.
1 153.
Radial section ?ridway between base and apex.
X 123.
Radial section through cortical region alone.
1 110.
Transaction of fibrovasoular region. X 857.
Surface view of needle showing stomatal
arrangement. X 348.
st --- stomatal process
go --- guard cell
so --- subsidiary cell
ss8 -- substomatal cavity
ep --- epidermis
hyp -- hypodermis
m --- esophyll
re --- resin canal
sh --- sheath
epi -- epithelium
en --- endodermis
tt --- transfusion tissue
sto -- strengthenlag cells
bp --- bordered pitf
ph -- phloem
p --- protoxylem
ma --- metaxylem
la --- lacuna
sp -- sieve plates
cp --- cortical plates
38
st
m1
,p
go an
"t
op
The tissue immediately within the dermal region is the
mesophyll, whose cells are large and thin-walled, and pos-
sess the highly infoMdedcondition which is so chnracteristie
of all pines. This infoldin- of the cell walls tends to
increase materially the inner wall surface against which
the chloroplasts lie. The most cons-icuous feature of the
mesophyllic region is the presence of resin ducts, waose
position in this species is internal, t.at is, they are
located adjacent to the endodermis. lhe resin duct is a
cavity lined by a layer of epithelial cells which are in
turn enveloped by a layer of thicker-walled cells which
support it. The ducts in this species generally number
from 3 to 5, although their position is more consistent
than their number.
The fibroyasoular or stellar region is separated from
the Aesophyll by an endodermis consisting of a single
layer of bead shaped cells. In this species the outer and
inner walls of the endodermis are uniformly thin, while the
radial walls are slightly thickened with suberin which is
recognized as the Caspariar thickening (Soar, 1922).
This species possesses two fibroyasc,:lr bundles wmich
tend to fuse together near the base and cpex of the needle;
yet in transactions taker. fro:, the iid-portion of t+e needle,
as in Plate V, figure 1, they are distinctly separate. They
are of the collateral type and are imbedded in a tissue of
thin-walled cells known as the transfusion tissue. This is
of a perioyclio nature (Takeda, 1915) and possesses con-
spicuously large bordered pits.
Lich bundle possesses a few cells of protox!,lem located
internally to the rest of the bundle, wtich re character-
istically small, round and weakly lignified. The cells of
metaxylem are larger, -enerally 3-5 angled, strongly lig-
nified, ana are in radial rows. Laoh bundle usually has
one or two rays which are conspicuous in the metuxyler be-
cause of the relative thinness of their cell walls, but
which fade out in the phloem. The aetaphloem is easily dis-
tinguished as being a group of thin-walled cells located
exterior to the xylea, and consisting only of sieve-tubes
and simple phloeu parenohyaa, yet no specific companion
cells have been detected. The protophloem is all but ob-
literated at the extreme outside of the bundle due to pres-
sure from the growth of the more recent elements within.
It is typical for the nature needle of this species to ex-
hibit one or two rather conspicuous lacunac ir that portion
of the transfusion tissue internal to the -rouips of proto-
xylem. -nother portion of the transfusion tissue consists
of thick-walled cells vwioh furnish su7-ort to t'e vascular
bundles. They occur in a linr adjacent to the phloem and
extend froi one vascular bundle to the other.
The above paragraphs have aeacribed the anatomical
features appearing in a transaction of a mature needle of
this species. Certain other noteworthy characters are better
exhibited in longitudinal sections through the sa-e tissues.
a radial section (Plate V, fi-ure 2) reveals tLe longitu-
dinal position of the Zuard cells. rhe mcsophyll appears
in transaction to be quite compact; however, radial and
tangential views produce a quite different impression of
the structure of this tissue. Radially sectioned mAterial
(Plate V, figure 2) reveals the mesophyll as a network of
filaments, each of two to three cells, extending .rom the
dermal region to the endoderais snd separated from each
other by intercellular spaces in as much volume as the
filaments themselves. radial section through the cortical
region alone, so that the mesophyll ,s shown extending unin-
terrupted from the dermal region on one surface to that on
the other, exposes these filaments to le in reality: the end-
views of plates of photoryntbetto tissue (Plate V, ftiure 5).
These pl-teo are separated by air Fnaces except whcre certain
cells of adjacent plates anastorose. T'.esc stite.ents are
clarified 1- figures 1, 2 and 3 of Plate V.
Another feature of longitudinal sections is te nature
of the thiokf ling of the various cell walls. These can
best be distia;uished in & radial se-tion (Plate V, figure
2). Apparently the radial walls of the endodermis, .hioh
contain the Gasparian thickening, are characterized by the
presence of minute simple pits. This view of the endodermal
cells shows also that the end-walls contain a slight amount
of lignin, especially at the angles.
The large bordered pits of the transfusion tissue are
conspicuous in radial sections. They are very frequent
on both radial and tangential walls and are without apparent
orderly arrangement. Radial sections reveal that the pro-
toxylem elements are characterized by spiral thickenings
while those of the metaxylem possess large bordered pits.
Similar sections show the sieve plates to be slanted toward
the vertical axis of the sieve tube. Another feature easily
observed here is the blunt end-walls of the thickened sup-
porting cells in the transfusion tissue. All of these
features are shown in figures 2 and 5 of Plate V.
HISTOGIMISIS OF THE NMEDLE.-
0haraoters of the apical meristem.-
Since the foliar primordium from which the pine needle
is derived arises from cells of the spur shoot it is well
to consider first the nature of this meristematio region.
The apioal meristem in the pine ".....consists of a group
of cells which may divide in any plane. A tunica is lack-
ing and the entire apex is regarded as corresponding to
the corpus of the angiosperms." (Korody, 1937). This state-
ment is corroborated by the present writer using material
from P. palustris. Figure 8 shows cell divisions in various
planes ana also the total lack of any well-defined zones of
aeristematie tissue. Such condition seems to be true of this
and other embers of the Abietaoeae (Strasburger, 1872;
Korody, 1997), while certain less closely related gymnosperas,
such as Taxodium (Cross, 1940), Qinkao (Foster, 1968) and
amia (Johnson, 1940) have shoot apioes characterized by
distinct zones of tissue with significant differences in
growth, planes of division, arrangement, and relative size
of component cells. Thus the unspecialized terminal meristem
of iPnus is a primitive condition and is an indication that
tM~s -roup antedates most other ryvnosnermous types.
The origin of all of
the anioal tissue of the
shoot can be traced ulti-
mately to the activity of
single groups of apioal
initial cells whioc divide
antiolinally, periolinally'
and obliquely without regu-
lar sequence.
Initiation tan eaerl
der loDment of ts a dl
priaordiua.-
Figure 8. Mear median The initiation of the
longitudinal section of
apical seristem of spur needle primordiua occurs on
shoot. X 400.
the side of the iosal merl
stem of the spur shoot, and is approximately 50 microns be-
low the submit. This lateral growth results from periolinal
divisions in the epidermal and hypodermal layers and also
from anticlinal divisions and radial elongation of certain
sub-hypodermal cells on the side of the shoot apex. The re-
sult of activity in these layers, as observed in figure 9,
is a definite outward thrust. In addition, certain cells
immediately above and below the region of cell division
have broadened the region of lateral growth by elongating
in a plane perpendicular to the surface of the shoot apex.
As growth proceeds there is organized a group of apical
and subapioal initials which continue to contribute new
cells by means of periolinal divisions. The region charac-
terized by this type of growth includes the adaxial surface
of the primordium, while the peripheral cells of the abaxial
surface proceed to divide antiolinally. Derivatives of the
sub-hypodermal cells add to the inner region of the pri-
mordiua by alternate radial elongation and anticlinal di-
visions. These features are expressed in figure 10.
The next stage illustrated (figure 11) is character-
ized by the marked elongation of the peripheral cells in
the distal portion of the primordium; by the continued anti-
olinal divisions of the peripheral cells in the nroximal
portion of the abaxial surface; and by the cells of the
medial region dividing antiolinally, periclinally and ob-
liquely without a regular pattern, yet vitl a predominance
of anticlinal divisions. Thus, the elongation of surface
cells in the distal portion and the continueA growth by
oell division alone in the more proximal regions result
in a gradual change in shape of the primordium from the
Irigove 9-1p..
Stooo of Initiation and sm1y 4eve1emnt
of the needle riaordium. 2"0.
rounded bulge as first exhibited during its initiation to
somewhat of a horn-like appendage. At this stage can be
distinguished also the elongating prooambial cells in the
peripheral layers of the shoot; however, these are not a
part of the foliar process but of the shoot axis.
As elongation continues the outline changes from the
horn-like appendage to a more linear form (figure 12) which
results from pressure on the inner surface by expansion of
the shoot apex and on the outer surface by the scales of
the bud. The internal characters expressed at this stage
are those of elongation in the apical and medial regions,
and also of intercalary growth near the base. The cellular
structure of the needle tip has bj this stage become fixed,
in that no new cells will be formed in the region. Here
the cells have attained maximum elongation and have com-
menced to vaouolate. The only other change remaining before
they mature is the thickening of the cell walls to various
degrees. Interoalary growth in the basal portion of the
needle primordium proceeds by the repeated insertion of
anticlinal walls to produce chains of -enetically related
cells. As the needle elongates, due to extension at the
base, it begins pushing its Aay through the overlapping
sales of the spur shoot. Only the epidermal and stelar
portions are represented in the primordium at this phase
of growth.
Origin of the cortical tissue.-
Now the initiation of the mother-cells which subse-
quently will produce the chlorenchyjatou; or cortical
region of the needle .ill be considered. The initiation
of this region does not occur until after the primordium
has attained a length of 300 microns. The changes which
occur during its origin can best be observed in transaction
(figure 15). Here the primordium is roughly deltoid in
outline. Most of the tissue at this level is characterized
by an embryonic appearance, because of the absence of im-
pressive enlargement and vaouolation, and also the pos-
session of prominent nuclei as well as the occurrence of
frequent oell divisions. The epidermal cells multiply by
numerous antiolinal divisions. The region beneath the epi-
dermis consists mostly of undifferentiated tissue and is
characterized by frequent cell divisions in various planes.
Hypodermal and sub-hypodermal initials arise at t.-is dtage
to form a layer two or three cells in thickness Just withinn
the epidermis, and from these the cortical tissue is derived.
a slight enlargement and distinctive staining reaction are
first noticed. Differentiation proceeds acropetally and
basipetally, until these initials extend froa Just above
the base nearly to the apex of the primordium. Their cell
division is periolinal in some places and oblique in others,
with the occurrence of a greater number of divisions in the
lateral corners of the primordiua than in the distinctly
adaxial and abaxial regions, thus resulting in the forma-
tion of the oortioal region of the young needle.
The origin of the resin duct mother-cells will now be
described since they are a part of this region. On exam-
ination of numerous sections of this particular region and
Figure 13. Transection of primordium 300
microns in length taken mid-way between
base and apex; Ia, epidermis; Jbh& hypo-
dermal and sub-hpoderal initials; iA,
stellar region. A 400.
stage it was observed there were normally from two to four
resin ducts initiated, with the latter number predominating.
Suoh usually oocurs in the lower medial portion of the needle
at a level quite above that in which the prooambial tissue
is differentiated. The canals located laterally seem to
precede the others in both time of initiation and rate of
development.
Resin canal mother-oells are initiated in the inner
zone of the cortical mother-oells. Their differentiation
becomes apparent because of a series of periolinal and
oblique divisions with subsequent enlargement of the de-
rivatives thus produced. Such conditions are expressed
in Plate VII, figure 1. Five or six mother-cells may be
distinguished in transeotion. At first they are joined
compactly to form a core around which another layer of
sells later differentiates; the inner core becomes the
epithelium and the surrounding layer develops into a pro-
tective sheath.
Increase jI thickness and initiation of prooambial
tissue.-
During the earlier stages of development the young
needle increases in thickness as a result of general cell
division through the central portion. Somewhat later an
additional increase in thickness is affected by cell di-
visions connected with the development of vascular tissue.
The initiation of prooambial tissue can usually be
discerned in the basal portion of the primordium when it
becomes approximately 225 microns in length. Unfortunately
no material which illustrates the first phase of vascular
tissue initiation was collected. The earliest stage ob-
tained was that of a well developed procambial strand, such
as illustrated in late VI, figure 1 (or 2). It is apparent
that the first-formed prooambial strand differentiates both
aoropetally and basipetally, which is in accordance with
Plate VI. Diagram (X 55) of a near median longitudinal
section of a young fascicle 3000 microns in
length showing fascicle scales and young needles;
also enlarged basal, mid- and apical portions
of one of the needles.
Figure 1. Basal portion including the spur shoot.
X 120.
Figure 2. Mid-portion. x 120.
Figure 3. Apical portion. I 120.
jsa --- spur shoot apex
M1 --- procambial strand
oan --- cortical mother cells
Am --- epidermis
= --- protoxylem
j= -- protophloem
tr --- transfusion
tissue
h=p --- hypodermis
A& -- apioal cell
AI --- outinized spur
50
VI
the development in ltave:; of the angiosperms. ihe proca-
bial cells divide periolinally and increase the \;icth of
the prooaiabial strand; conbequentl;, this results in the
thilkening of the ,oun,; needle.
Intercalary elon,'ation of the needle.-
Growth by apioal and subapicae initials characterizes
the early stages of primordial devolopnaent anc is respon-
sibl: for only a small portion of needle elongation. The
apioal initials account merely for the extreme tip, while e
the subapical initials divide more profusel- and their de-
rivatives are responsible chiefl:- for the portion n abutting
the tip (Plate VI, figure 5). They are early maturing
regions which contribute little to the incre. e in len-th
of the needle. subsequent needle elongation is due pri-
marily to intercalary divisions and to t.ah e-xtenaijon of
epidermal and conductive cells.
The interoalary activity, us previously rantioned, ooours
in the basal portion of the needle and proceeds by the re-
peated insertion of anticlinal walls to produce vertical
chains o.,' genetically related cells. Such affects a much
greater increase in length than in width, thereby resulting
in the characteristic linear form of the leaf, as for ex-
ample, a young needle, as illustrated in Plate VI, may
have a length of 8000 microns, yet this same needle (Plate
Vil, figure 1) has a width of only 300 microns.
The figures in Plate VI represent sections taken at
various levels from a longitudinal plane of the needle.
In the upper portion of figure 2 of this plate can be ob-
served the initial intercellular spaces connected with
the differentiation of the chlorenchyma. Juoh cre first
formed about 500 microns back of tha tip, :;d such activity
proceeds gradually toward the base. Here the conductive
tissue is well differentiated (Plate VI, figure 2), as
evidenced by the appearance of spiral thickenin's of the
protoxyle elements as well as of sievc plates in the phloem
region, and also of simple pits in the endodermis.
The cells in the base of the .o ;xi; needle are meris-
tematic ano shortt rows of their derivatives may be observed
in figure 1 of Flate VI. Growth continues by repeated
division in these chains of oello, resultin.- in the elon-
gation of the needle. ,eristenatic tissue formed at the
bae of a young needle is composed of vertical chains of
cells which divide in a plane perpendicular to the main
axis, and is termed the "rib meristea" (eohiepp, 1926).
The maitotio activity extends much farther in the cortical
region than in the conductive region. A majority of the
cells in tbh latter region cease dividing and oo:imenoe to
elongate at about 500 microns from the b&se. Hence, ver-
tical growth, subsequent to the earliest stages, results
from the activity of the rib meristem and the enlargement
of its derivatives together with elongation of the cells
in the ,rovascular region, and also of those of the rein:
canal. rhe region of maturity in tlt apicul portion be-
sins tomovr toward the base al the needle cxte:nds, so that
in later growthh the rib merist.- is found only in the ex-
treme basal portion. The epidermis parallels t' is internal
increase in length by 'ell Oivisio-: and eel. enlnr-ement.
Differentiation and devi lop-ent Lrs seen ein crial
transeotions.-
Evider,t for tl.e ,reviou- satf'e:aent.~ supports the
fact that pine needle develo-ent,: is c2.araotcri.t:d b.-
linear growth in wLioh the aeristeautio activity ,nd elon-
gation proceed acropetally from thu buse anu uLturation
extends basipetally from the tip. Therefore s;rial trans-
verse sections front base to tip of young needles are the
best means of studying tissue differentiation ,nd growth
to maturity, so that from several series of tra~sverse
sections of young growing needles certain sci.utionsu .ere
made to represent the status shown in Plates VII-1 in-
elusively.
The first stae (Plate VII, figure 1) in this series
represents a transaction taken at about .04 of a millimeter
above the base of a needle which is 25 millimeters in
length. Sections immediately: beneath it reveal the apex
of the spur shoot between the bases of the three needles
of the fascicle. The vascular elements here are already
well differentiated as is borne out by the appearance of
elements of protoxylen and protophloen on either side of
groups of undifferentiated procanmbial tissue. 'he vascular
bundles it this level are still onnected both aove and
belo:. A band of protox-lcmr elcientr xtendt xtn ct':een the
two bundles on the .daxial sidc, :':ile a similar ocnneotion
is mede across the abuxiul rc-ion ty clernnts of :rotophloea.
A fe.. cells of transfusion tissue, which are not icriate;atic
in appearance and which, by their distinctive staining re-
action, enable the bundles to be distinguished fror each
other, lie between the groups of procarbium.
The other reci-ns here are still neristeritic. The
groups of resin canal mother-cells which will produce a
lateral re-in ocnal may be distinguished in the riCht-hund
corner of this trunsection. The cells of the ejidermis,
although easily distinguished from the adjacent tissue, are
undergoing numerous anticlinal divisions There is evi-
dence that the cortical mother-cells nre beginning to dif-
ferentiate. Certain cells in the second layer beneath the
epider-is appear to have undergone chemicr1 chsnger which
cause them *o toake the distinctive rtainin- re'.ction so
characteristic of cortical tissue in the pine needle. lIse-
where in this section the cells aaz he observed to divide
in various planes. Those nearest t.e vascular bundles mul-
tiply by the insertion of walls in a plane tangential to the
surface of the bundle. Immediately outside of this region,
especially in the lateral areas, ell division is oharacterised
Plate VII. Transeotions of young needles representing
the first three stages to trace tissue dif-
ferentiation.
Figure 1. (Stage 1) Transection .04m. above base of
needle 25-50mm. long.
Figure 2. (Stage 2) Transection .06mm. above base
of needle 15-20mm. long.
Figure 3. (Stage 3) Transeotion .15mm. above base
of same needle.
All figures X 120.
pz --- protozylem
po --- prooaambium
pph -- protophloeo
rom -- resin oanal
mother-cells
one --- cortical mother-
cells
ep --- epidermis
hyp -- hypodermic
on --- endodermis
trt --- transfusion tissue
yX
by the insertion of both tangential and radial walls ith-
out any definite pattern. Division in the oortimal atheW
*sel layw, .ad also in the layer botwmn t i a t he *l*
O *Jm in eolusively atl elinal, e*z I 1 t he eera
wW w It Is ipoelolal and a1so obliquo
ft o-a stage Is i llu tra te& Io 1at* eT M 1
eto. e)-rs me, -"---*
Owe.SI~ I~S14)~
.I~~ .. ~
V .r -.
&. .,
.6i hrunt@ ~rr
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,' .
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amt.~~~~a "pboAtts ab tm aV r"~4~ ou"
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'Zn. ~UI
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tiuae iffrttertlatitea
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slrase a* a ws 5)
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serattions so sheraeteristio of the angles on the adazial
smrfaoe of pine needles occurs as an out-growth of the epi-
dermal ell in the right-hand corner of the section ea-
ployed. The most notable changes exhibited in this stage
are those resulting in the expansion of the vascular bundle
and the differentiation of stonatal nother-sells in the
epidermis.
The fifth stage (Plate VIII, figure 8) is from a section
out one and three-quarters milliasters above the base of a
enodle twenty-five to thirty all.laeters in length. The
aetively dividing proosabium has been forced outward by
the rapid enlargement of the immature metaxylen sells.
Three stages of metaxylem development can be differentiated
here. Mature etaxylem elements with lignified walls lie
adjacent to those of the protozylea. Younger metaxylea
elements border the mature elements and are approximately
the same size and shape, differing only in the presence
of cellulose in their thbikened walls instead of lignin.
The youngest metaxylne elements are large, thin-walled and
abutting the proeaabium. laoh vascular bundle is still
Zpeanding by the process previously described.
Sth eoatinued enlargement of the cells in the trans-
faLea and eortioal tissues results in an increase of the
dieaolems in eross.seetion. The endodermal sells are tflly
&a l.are as thoae of the adjacent aesophyll, but iffter is
their *etest as they take so staln. eo thiekesnin ef the
radial walls in this layer is evident at this stage. The
sheath oells associated with resin ducts are clearing up
so that few possess substances which reaot much with the
stain. On the contrary, the secretary cells are still
functional as such, as indicated by their prominent pro-
toplasts, and in addition the canal becomes more prominent.
Most of the cells of the inner layer of hypodermis have
cleared up because their contents do not stain. Suoh is
not the case of the sells in the outer layer of the same
tissue, since their contents stain brilliantly. The con-
tents of the typical epidermal polls in general have lost
their former staining property, yet the stomatal mother-
sells still posesss it. The central cell in each group of
the latter has further enlarged, most of the expansion oe-
surring inwardly so that the two hypodermal cells immediately
beneath are being forced apart.
The next stage (Plate VIII, figure 3) represents a seo-
tion taken two and three-tenths millimeters above the base
of the same needle employed in the preceding. The outstand-
ing features of this phase occur in the vascular and dermal
regions. More of the younger differentiated metaxylen oells
have acquired a cellulose thickening, while those of the same
stage in the previous phase have now become lignified. A
premient feature of the vascular bundle at this stage is
the appearance ef definite rays of oells extending from the
yenagest etastyle aerosa the undifferentiated vascular
tissue into that of the youngest phloes. The rays cells are
more isodiametrio than those on either side, and where they
ocour in the xylem or phloem their brilliantly stained proto-
plasts completely fill the cells. The endodermal sells have
become less angular than previously. Certain stomatal mother-
cell groups have changed considerably since the central cell
of each group has enlarged and, because of the pressure from
the adjacent cells, has been forced for a short distance
beneath the epidermis. Several of these depressed cells
have divided antiolinally so that now there are two sets of
cells to each group, namely, the pair which were on each
side of the central cell originally and are still on the
same level with the epidermis, and also the two new cells
resulting from the division of the central cell. The latter
pair, as they enlarge, act as a wedge and force the hypo-
dermal sells apart. The lateral sells of the stomatal group
become the subsidiary cells, while the derivatives of the
central sell are the future guard cells. Suoh a combination
forms the mature stomatal process. Subsequent stages in
its development will be described presently.
The three following stages (Plate IX, figures 1, 8 and
5) were obtained at three and one-half, four and one-fourth
and 12 millimeters respectively above the base of the same
needle. They express later phases in the progressive develop-
ment of the various tissues and processes whose different-
ation has already been described. The remaining prosaebiun
gradually ceases meristematic activity and its cells enlarge
preparatory to the final differentiation into xylem and phloem.
Further lignification occurs in the xylem elements, and the
cellulose walls of the phloem cells thicken to some extent.
The transfusion tissue increases in area by Means of a pro-
nounced radial elongation of the cells located adaxially
and abaxially to the vascular bundles. Bordered pits now
appear in the thickened walls of the transfusion cells as
simple pits which signifies their present immature state.
The increase in space occupied by the central cylinder re-
sults in the stretching of the endodermis in a tangential
direction so that each cell generally assumes a more ellip-
tical shape. The chlorenchyma gradually attains its charac-
teristic, unfolded condition due to the many invarinations
of the cell walls. The epithelium of the resin duct becomes
somewhat compressed as it stretches to compensate for the
increase in size of the canal. The sheath about the secre-
tory structure also extends and the initial increase in
sell wall thickness occurs. The most conspicuous develop-
ment in these three phases is that of the stomatal pro-
cess. The enlargement of the subsidiary cells forces the
guard cells deeper beneath the epidermis until they appear
as actually a part of the hypodermis. The sub-stomatal
cavity makes its initial appearance as a result of the
opening of one of the invaginations of the adjacent cortical
cell as the guard cells diverge. The subsidiary cell walls
Plate II, Transeotions of young needles representing
stages 7, 8 and 9 in the series to traoe tissue
differentiate on.
Figure 1.
Figure 2.
Figure 3.
(Stage 7) Transeation 3.5ma. above base
of needle 25-53am. long.
(Stage 8) Transeotion 4.25mm. above base
of same needle.
(Stage 9) Transeotion 18em. above base of
same needle.
All figures 1 180.
P A-
oo
44 4- Wo
Ilk(
A&L
thicken slightly, especially bordering the outer vestibule,
and also next to the leaf surface. Walls of the epidermal
cells thicken to some extent and cutinization begins by pro-
ducinr: a thin film on the surface of the leaf which thickens
on the beak of the outer vestibule of each stoma. The guard
cells become small and elliptical, and produce the callose
thickening characteristic of their mature condition in pine
needles. The hypodermal cells enlarge, attain their maximum
size and thicken slightly. The last of these stages occurs
at about the level of emergence from the fascicle sheath.
During the final stages (Plate X, figures 1, 2 and 3)
in this series the developments Just described continue to
their culmination in the mature needle structure. Such
changes include: The development of pits in the metaxylem
walls; the thickening of strengthening cells in the trans-
fusion tissue adjacent to and connecting the phloem of the
vascular bundles; the maturity of the bordered pits in the
transfusion tissue; the compression of the epithelium against
the encompassing sheath and the slight thickening of the
sheath cell walls; the suberization of the radial walls of
the endodermls resulting in the Casparian thickening; the
increase in the cllosc thickening of the guard cells and
in the cutinization of the beak bordering the entrance to
the outer vestibule of the stomatal process; also the ex-
cessive increase of thickening in the epidermal cells almost
to the point of obscuring the cell cavity; and finally, a
Plate X. Transections of young needles representing the
final three stages of the series to trace tissue
differentiation.
Figure 1. (Stage 10) Transeotion 17.7mm. above base
of needle 25-30mm. long*
Figure 2. (Stage 11) Transeotion 19.3mm. above base
of the same needle.
Figure 3. (Stage 12) Transection 30mm. above base
of needle 60*p. long.
All figures 1 120.
aimila thickening of the inner hy odornal cells as well
FIs an increase in thickness of the outioular layer. There-
fore the final st:-cy in t.1is series (Plate :, figure 3)
possesses fully developed Atructures which, with one ex-
oeption, are quite similar to those in Plate V, figure 1.
This exception is the .rest difference between the
number of xylen and phloe'- elements occurrin- at the sane
level in a more nature leaf. 'he section of "i-ure 3 in
Plate X is taken fro.. the Alid-recion of a needl-e 60 -lilli-
,leters in length, and c .oh vusczlar -:-ndlE contains t'Lree
or four protox;-le.i and a provi.u.tel. 12 .t..x;yl:', -.lerents.
. section necr tihe base o the saLe needle r.vcals. fro:.
five to seven protoxyle.a and Cbout 30 mletaxylt:M elements.
This pronounced increase in number of aetaxyle:a elements
in the latter section is readily attributed to its lower
position in the needle. Tihe number or phloea elements in-
creases in the same proportion. Similarly, the base of a
needle of ,aaximum length (300 to 3b0 millimeters) has a
;iuch Lrestor number of vascular elements than that which
occur; in the mid-portion which is substantiated by oom-
parins figure 14 with figure 1 of Plate V. In fact, the
base of a fully nature needle is conposed mostl. of the
vascular elements. The vascular tissue of this region has
a dual function in that both conduction nnd mechanical sup-
port are performed b- it.
70 .
Figure 14. Transeotion through basal portion
of needle 320 millimeters in length. 1 70.
Further features of development.-
Certain relationships exhibited during the development
of the various regions to maturity are more conveniently ob-
served in longitudinal sections where any oell elongation
oan be detected. rigures 15 to 20 are from a needle of three
and one-half millimeters in length, and show progressive de-
velopment in the longitudinal plane. The base of the needle,
as represented by figure 15, contains the meristematio region
from which the tissues differentiate. The procambial strand
extends downward and is connected with the vascular elements
of the spur shoot. The strand extends upward into the
ai N'_410
differentiating foliar tissue above the base of the needle
and first differentiated into the protophloen centriCugally
and later into protoxylem cntripetally. The re-:imining cells
illustrated in thio ^iure are uefly ;~r oriste::utic tis-
sue.
The oortical motht-r-cells can e~rly; b reco nized some-
what above the immediate ba.L (figure 16) as indic_,t. by
their c.haracteriatic stainirn reaction. The resin canal
:aother-cells are obcsrved Lt Lbout the sa.ee l.vel hadC ,re
identified by the absence of the deep stainr of tht cortioal
mother-3ells and yet are of the sa;_ Len era:lu shape, vw-.:h
distin.uishfs t em fro~ the r&aidly elonCating cells of the
stolar region. liere the ejiderimal cells are easily actooted
as they retain their iaeristeatic activity, multiplying by
anticlinal division~. :he stelar tissue. (ifferentiate
(figure 17) first by jroducin5 phloem from the outer portion
of the procambial straad anc then,at a slightl: higher level,
xylei elements from the inner portion. The latter have the
chtractcristic spiral thick.nin s. The central portion of
the procauabial satind rerLains undifferentiated at this
level. The es:in ctaii.l itLslf appear_ a:iong the citahlial
cells "t tAis level, .nd t.L shicatii cells _avw undcr;one a
decided elongaution. 2he cpi.erala cells have ceuded dividing
in a plane perpendicular to tle Aain axis and have elongated
to soue extent. The hypodermal cells have Llongated to even
a greater degree and are thus distinct frou the cortical cells,
although the contents of both appear very muoh alike.
Figures 15-20. Diagram (X 75) of
section of needle
including apex of
with enlargements
various levels to
of development.
sea --- spur shoot apex
ps --- procambial strand
ep --- epidermis
omo --- cortical mother-cella
rmo --- resin canal mother-
cells
pph --- protophloem
px --- protoxylem
near median longitudinal
3000 microns in length
spur shoot at the base,
(I 144) of portions at
illustrate further features
po
ec
ro
smo
oo
so
go
op
--- procambium
--- epithelial cell
--- resin canal
--- stomatal mother-
cells
--- cortical eells
--- subsidiary cells
--- guard cells
--- cortical plates
72
*, '- 'q "
*4.* & ,.. *
.Rt.
P1o
,
-".
*8 .-'~
V 6
'I:,
t1
'a *
I.. .
>.
* .
Vt.-
.1:'
ti
' Iv
* I'
Lu
I. .
Se-
44
The invagination of the oortioal cells makes its initial
ai j,-...rtnce above the )reoeCin- figure, as shown in figure 18.
Viewed froa this Ilanc each forerly cutical oell acquires
an infoldin: of the 'vall on ecc1 side ,whic'- (-ter-d!- cross
so Ps to divide it horizontally along a trqnsv~rse line.
Another development occurrin- in t+his -c-ir;n 1 *'e initi-
ation of the rtomiatal mot'.er-cells ;:: Indicated h:- tlh ,light
enlargement of the ot"trl ele:;ant of ,3ac>. arou'' n: its sub-
sequent indentation of the surface.
Thu irtercellular 3 ases assoctted vith the approaching
maturity of the cortical cells first app(.&r at t. point in
the leaf as indic ted' in fir'ur'e 19. They are produced when
the cells of tht Cera.l ~.:c stelar region undergo such ex-
trene elonegtion as to cruse unequal stresses in the cortical
region resulting in the cleavage of thr. latter cells along
transverse lines. Further -tretchinc extends them so that
at maturity th e hlorenchyma occur, as trunsvers.e plates
between the derual .n. stellar regions and is separated by
interoellular spaces, except where adjacent plates anastomose
(figure 20).
This cocountt for thr condition exist.inr in the ohloren-
ohyma as it appears In a longiti.dinre] ectirn. IIHoever, the
peculiar infoldine of the mature cortice.l cells as observed
in needle transeotions can bt explained b;- in exerination of
the figures in Plates IX and X, in which one may first notice
these invaginations at about the time the endodermis, hypo-
derais and epidermis are approaehing maturity. The latter
may mature in advance of the cortical tissue, and their walls
br~ooe inelastic. As a result of ti9s confined nrea about
the cortic~l cells, and due to the hil;h degree of rissllenoe
of their walls, marked convol':tions occur.
Comparison .4th a ,aiseDao. .-
The developmental processes of the pine needle, as re-
veale6 in tViis investigation, uu-Zest several features that
are in common with an-iospermous leaves. Increase in length
results fro:. the combined activities of npical, subapical,
and intercalary growth. The apical and subapioal initials
mature early and subsequent elonW-tion proceeds by inter-
oalary cellular division and expansion. The rib ueristem
characteristic of t.is leaf is found in angiosperrs in the
petiole and aid-vein alone, while the :neaophyll in anslo-
sperms (Foster, 1936) Zenerclly originr.tes from a plste
meristem rather than the former. .v.nong the anirosprm? the
derivatives of the sub-mar-inrl initial:e forr, a Nlnte
meristem whichh results in tMe broadl- lateral oro'rth of the
la.ina and thereby differentiation is nade between petiole
and blade. However, the sub-marr:inal initials in the "ine
needle become the cortical mother-cells by the formation of
a layer uniformly" of t-.o to three cells, with no lateral
expansion into a blade. Such is largely responsible for
the linear fori of the mature needle, and this, together
with the organization of a highly active rib meriatem, so-
sounat for the attainment of a length very nuoh out of pro-
portion to its restricted width.
The activity of thb rib merietem in rrcducing vertical
ohains of '~etcally rel.tcd cclls End the .tresscs 'ro-
duced by the clongatin. derr.l -nc' stelar elements cre
rem onsible for th1 ;.roduotion of the first intercellular
spac-s in the cortical region. countss (1932), very (1935)
and Poster (19"6), Rnonf others, mention r. sililnr plcno-
nenon ir enCiorperms. louwever, the stresses ir, -r,.-iospermous
Icr.ve: ure lateral &s -,ll i.; verticO l because of the 1.o-
tivitl of the plate :.eriste.i i.nd th(t .ubsecuent lr.tc.r&l as
well os verticac enlt.rcfnent so thnt the cuclls of the p,.li-
s:?dic region r~ceparute ir. ny direction ;.w.rE.lle3 with tle
surface of the Icr.f. The flnrl developrcnt. of tr IntPr-
celluler spi.ces ir thr ?inr rneeder restrltr fro- thr con-
tinued clontloz! of the rrr-r n3 *nd 'te]lir elcrients. Since
this is .:r1r(1 r ity J Icr ituc'r rti r f t.r rperdle,
ti(: ctr(:sLes,. zre cdircctcd v(rti-c12l3y, :.n; thr intercelluler
spaces urt. corresbonrinl:- in t l.orizonttl r1 lne.
.n appreciation of th( aiEnificance of thcsc histo-
genctic features as beirn; indicative of ,hylor:ei,-tic re-
lationships must :..it the results of si..ilLr studies con-
oernin4 ot: :r Ly:.i-ospcri.s, in particular tho&e 'ith broader
leaves, such s .: rauoeria.
sUat&AlY
The vegetative buds and needles of swamp pine (k.
palustris Mill.) were employed in this atudy. they were
collected in progressive stages of development from
saplings and adult trees in the vicinity of the campus of
the University of Florida during the growing season of
1940-41. External growth measurements were made to sup-
plement the histogenetio data by determining the general
region of needle elongation.
Standard histological techniques, with certain modi-
fications, were applied in the preparation of the material
for microsoopio study.
The needle primordium is initiated laterally from the
apioal meristem of the spur shoot. Only the outer layers
of the apioal meristem are involved.
Growth of the primordium proceeds by apical and inter-
oalary activity until the tip matures; tLis is followed by
intercalary growth alone.
Only the epidermal and Atelar portions are represented
during the earlier phases of growth. Sub-marginal initials
which become the cortical mother-oells do not appear until
the primordium is relatively well advanced.
Serial transactions from the base upward of young
needles reveal tissue differentiation in the following
general sequence: prooambiua; protoxylem and protophloes;
epidermis; resin canal mother-cells; oortical mother-ooells
transfusion tissue; epithelium and cells of the encom-
pascin; sheath; hyrcder::is nd i.eso;hyll; metaxyle. c..nd
metarhlot:; endodermis; stc:.-ital mother-cells; u&rrd and
subsidiary cell. However, the tiscuLs mature at different
rates.
The highly invasTirted mesohtyll results fro:, the
resilience of their w-lle cnd from the restricted .res
e:nlosed by the retraining: .-alls of the rerzal and endo-
dermal layers.
The intercellular srccs occur in th: m sohtyll -s a
result of vertic-l dtr.essc rM ct~c-Crr.t th cells rlong
horizontal lines, thereby .'or-ine trc-svwrsec-l"tes .. tissue
extending ttv.eer, the =ndcT.r-iL ..nd typoder.r.is.
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