Ruhher and Iatex.

By
Barbarin Arreguin.

lntroduction.
Rubber has been utilized in the manufacture of many goods, at the present
it is considered to be one of the most essential raw materials. Natural rubber
is produced by the plant kingdom, and it is found distributed in about two
thousand species of plants belanging to the dicotyledons. The plant families
which areprominent as rubber bearers are: Euphorbiaceae, Moraceae, Apocynaceae,
Asclepiadaceae and Compositae. A historical review on the rubber in Russia
hy PIOTROVSKI covers the 18th and firsthalf of the 19th century. The literature
has been weil compiled up to 1947 in a review by BoNNER and GALSTON.
Any study on the origin and biosynthesis of rubber and related hydrocarbons
by plants, will enable us to understand better the physiology and biochemistry
of these secondary but important plant substances. In spite of the many publica-
tions on the technology of natural rubber and its multiple applications, little
has been published on its biosynthesis. Among the many difficulties met in
this kind of studies, it is worth mentioning the slow rate at which the hydro-
carbon is synthesized by plants. Even at the present time where synthetic
rubber has achieved great improvements in quantity and quality, natural rubber
holds still a unique place in world consumption.
Rubber is produced by tropical and temperate zone plants and it is stored
in the upper and underground portions of the plants. The most important
rubber plant producers are Hevea brasiliensis, a native plant from the Western
Hemisphere, Parthenium argentatum, Taraxacum kok saghyz, and Cryptostegia
grandiflora. Good general reviews are those by BoNNER and GALSTON on guayule,
on Taraxacum kok saghyz by WHALEY and BowEN, on Cryptostegia by STEWART,
BaNNER and HuMNER, and also the textbook by BoNNER.
Up to now the main source of natural rubber has been Hevea brasiliensis.
During the last two decades there have been intensive studies on the possibility
of growing commercially such rubber containing plants as Cryptostegia grandi-
flora, Parthenium argentatum and Taraxacum kok saghyz. Throughout this
article when the term rubber alone is used it is referred to natural rubber.
In table l taken from METCALF (see VAN RossEM), the yields of various
ruhber producing plants are shown:
Table I. Y ields of rubber producing plants in lbsfacre.

Hevea brasiliensis. High yielding clones. . 2,000

Guayule. Partheniurn argentaturn in the US 185
Taraxacum kok saghyz in Russia . . . . 106
Taraxacum kok saghyz in the US . . . . lOS

The figures of table 1 indicate that Hevea is by far the best rubber producer,
however it has been stated by NrsHIMURA, HIROSAWA, and EMERSON that guayule
ruhher wonld make a good Rnhst,itute for HevNJ, plantation ruhher. Jt has heen
B. A. Lozano et al., Der Stoffwechsel Sekundärer Pflanzenstoffe / The Metabolism of Secondary Plant Products
© Springer-Verlag Berlin Heidelberg 1958
224 BARDARIN ARRF.GUIN: Rubber and Iatex.

repeatedly reported that guayule under irrigation and favorable soil and climatic
conditions, may produce as much or more rubber than Hevea. The yields of
Hevea have contin1:;ously increased owing to seedling selection and vegetative
propagation of the best material. It was concluded by MAAs that twelve hundred
lbs. per acre per year of rubber are common yields in Hevea, and that monoclones
produce easily eighteen hundred lbs; he also indicated that material yielding
less than the latter figure should not be used now for agricultural purposes.
Indeed this astanishing progress has increased four fold the yields of thirty
years ago. The scientific work carried out in Malaya, Java and Sumatra has
permitted 30 to 40% of the cultivated areas of these countries to be put under
cultivation with high yielding clones.

Latex.
Rubber is produced primarily in the stem andfor roots of rubber bearing
plants in the form of latex which consists of microscopic particles of rubber
suspended in a serum. Fresh undiluted latex is a very complex colloid. Looking
under the microscope, HAAN-HOMANS, DALFSEN and VAN GILS found that besides
the rubber globules the latex contained much larger irregular colloid particles
named lutoids. Ammonia dissolves the lutoids, thus this solvent ha'3 prevented
the observation of lutoids since it has been a common practice to add ammonia
to fresh Iatex to preserve it. If latex is found in a plant it does not necessarily
indicate that rubber is present therein since particles other than rubber may
be in suspension.
The latex particles vary in shape and size (see HENDRICKS, WILDMAN and
McMuRDIE), their diameter fluctuates from 0.01 to 50 microns. Some common
forms of the rubber particles are pear shaped as in Hevea, rod shape:l as in Manihot
and spherical as in kok saghyz. The latex is found in living cells and vessels, within
which the rubber particles in active Brownean movement are distributed. In
some plants rubber is confined to specialized cells as in guayule, whereas in
others the latex is located in especial vessels, from which it flows out after tapping.
In order to get the latex from the lactiferous cells the cellular structure has to
be broken by milling combined sometimes with heat treatment.
To the planter and manufacturer the quality and the mechanical and chemical
stability of the latex are very important properties. The first is measured by
mechanical agitation and the second one by establishing its stability after the
addition of ions. Furthermore the electrical charge that surrounds the rubber
particles has a profound relation to the stability of latex. It is known that the
electrophoretic mobility is a function of the electrical charge. In colloids the
electrophoretic mobility is a good method to determine their electrical charge.
BowLER studying fresh diluted latex kept at constant PH by the use of adequate
buffers, showed that the isoelectric point differed somehow from clone to clone
in Hevea, as well as from tree to tree of the same clone. The isoelectric variation
ranged from PH 4.04 to PH 4.62 indicating different surface charges. The addition
of ionic substances caused an initial decrease in latex mobility, this effect tapered
off at higher ionic strengths. A straight line was obtained by plotting the logarithm
of ionic strength versus the mobility. Ammonia preserved latex showed variation
of the mobility with aging, producing increased mobility after 30 to 90 days.
This effect is attributed to the hydrolysis of esters of fatty acids of high molecular
weight which are normally present in the latex, and that once hydrolyzed formed
ammonium soaps, which either displaced the proteins from the rubber particles,
or formed a complex with them. At lower PR the increased mobility with aging
is less noticeable. It has been mentioned that the serum constit,uents of the
 Latex. 225

Iatex will affect the mechanical stability of rubber by changing the d.istance
of the electrical double layer, without affecting appreciably the net particle size.
ALTMAN (l to 4) has developed a general scheme of latex analysis, which
essentially consists of treating the latex with various solvents like ethanol,
ether, water etc. In this manner the rubber is purified, obtaining the proteins,
acids and Iecithins separated in other fractions which then are analyzed. This
method in general may be applied to any plant material containing Iatex and
in the case of Hevea Iatex the following values have been obtained which are
tabulated in table 2.
Fractions land 2 combined contained Table 2. Analysis of fresh Hevea latex.
in add.ition to rubber and proteins 0.12 mg No.j Fraction I Values in %
of copper/100 gm. Fraction 3 included
amines, amides and amino acids and 1 I Rubber hydrocarbon. 36.90
little ash. Fraction 4 was made by fats, 2 Protein . . . . 1.03
Iecithins, cephalins, organic acids, glycerol 3 Water soluble. 3.20
4 Ether soluble . 0.9-1.1
and some ash, and non-saponifiable sub- 5 Solids . . . . 0.30
stances. 5 contained prolamines and sterol-
ines, finally 6 contained some phosphates,
6
7
Precipitate . . .
I
Total solids in latex 42.4-42.6
0.10

raw proteins and little rubber.

VAN DEN TEMPEL (1, 2) studied the acidic components of preserved Hevea
Iatex, which were identified partly as volatile fatty acids and higher fatty acids;
other anions present were phosphate and carbonate. It appeared also that
the addition of ammonia reduced the formation of volatile fatty acids, whereas
the fatty acids of high molecular weight increased with time.
If fresh Iatex from Hevea is adjusted to PH 7.0 and centrifuged, two layers
separate: a yellow fraction at the bottom and a white fraction above. Careful
analysis of these two
fractions were carried Table 3. Analysis of the white and yellow fractions.
out by HAAN-HOMANS, Original White Yellow
VAN DALFSEN, and VAN I Iatex fraction
I fraction

GILs (see VAN RossEM).

Weight ratio 100.00 76.3 23.7
The results of these Specific gravity . 0.977 0.966 1.0016-1.0051
analyses are found in Viscosity in centipoises 11.7 9.2 -
table 3. Dry ruhher content % 34.8 43.7 9.0
From table 3 it Total solids % 37.4 45.8 14.3
Nitrogen content % 0.24 0.22 0.33
readily can be seen that Ash content % 0.60 0.47 1.11
the yellow fraction con- PR fresh 6.3 6.9 5.7
tains some of the none-
rubber components of the Iatex, including the greatest inorganic fraction. This
portion serves to stabilize the whole colloidal system. The bulk of the rubber
is present in the white fraction which contains the major part of the solids.
Other workers obtained four fractions when fresh Hevea Iatex was centrifuged
at 59,000 X g. These four fractions obtained by CooK and SEKAR were at the
top a fraction 1) consisting of a thick cream layer of rubber particles, 2) immed-
iately below an orange yellow layer of globules, 3) a clear serum and 4) a lutoid
fraction at the bottom. Fraction 1 contained 70-80% solids and made up 60%
of the whole Iatex, fraction 2 consisted of globules similar to those previously
described by FREY-WYSSLING in Hevea brasiliensis, H. guianensis, H. colina,
and H. spruceana. The globules of fraction 2 were made up of colourless resin
globules which carry the yellow pigment of Iatex, and are of the same order of
magnitude as the rubber particles. They refract light more than rubber, have
a buttery consistency, cohere better than rubber and, they can be stained by
Handbuch d. Pflanzenphysiologie, Bd. X. 15
226 BARBARIN ARREGUiN: Rubber and latex.

fat colouring dyes. The components identified in this fraction 2 were various
esters of fatty acids; these are liquefied by formaldehyde, they reduce silver
nitrate, osmic acid, and are oxidized by potassium permanganate and nitric
acid. The formation of resin globules in the Iatex is concomitant with the forma-
tion of the ruhher particles by the Hevea tree. Fraction 3 consisted of a pale
brown viscous liquid containing 7% solids and formed about 25% vjv of the
entire Iatex. This fraction contained most of the protein and mineral salts of P,
K, Ca and part of the Mg of the Iatex. Fraction 4 was a gelatinous grey yellow
mixture, containing 12% of solids which made up 12-15% vjv of the whole
Iatex. By digestive treatment with ammonium hydroxide it became partly
soluble, producing an insoluble precipitate of MgNH4 P0 4 , probably identical
with that described by McCoLM, which consisted largely of an insoluble precipitate
of MgNH4 P0 4 • 6H 2 0. The last fraction had packed transparent islands re-
sembling the lutoid fraction of HAAN-HOMANS and VAN GILS also, described by
HAAN-HOMANS, VAN DALFSEN and VAN GILs. Likewise this fraction contained
most of the magnesium.
Experiments with mature seedling trees by PHILPOTT and WESTGARTH showed
that fertilizers had a pronounced effect upon the stability of ammonia stabilized
Iatex. Utilizing K, P, and Mg fertilizers, they found that low stability is accom-
panied with low K and P, and high Mg. Latex from a single area was used,
but the evaluation of clonal Iatex is complicated by secondary factors. It is
known that some latexes obtained from particular clones show very high and
unusual stability. The explanation offered here was based on theoretical and
experimental data, which indicated that a low P/Mg ratio conducive to low
stabilitywas caused by an interaction between P andMg ions, existing conducently-
insufficient P to remove all the Mg. Conversely, excess of P ions destabilizes
the Iatex since the surplus P interacts with charges on the surface of the Iatex
particles. If P and Mg were present only as ions, the weight ratio for complete
precipitation as MgNH4P04 would be 1.35. Hence latexes with a PJMg ratio
either less or greater than some optimum value would be unstable. The observa-
tion on six clonal latexes of which three were abnormally stable showed that
these had a PJMg ratio either less than one or higher than two, whereas the other
three whose stability was high had a ratio between one and two.
The spontaneous coagulation of Iatex which was thought to be caused by
acid and brought about by bacterial action, now has been shown by v AN GILS
(see VAN RossEM), to be soap coagulation. This coagulation could be induced
by the addition of sodium salts of fatty acids. When soap is added to Iatex
the fatty acid ions are strongly adsorbed on the ruhher particles causing a dis-
placement of the protein layer, which becomes unstable in the presence of Mg or
Ca ions due to coagulation. Consequently the coagulations are dependent on
the presence of Mg and Ca ions in the Iatex.
The non-ruhher constituents of various latexes have been reported in many
publications. The work of ALTMAN (1 to 5) is very significant for Hevea Iatex
and that of STEWART, BüNNER and HUMNER for Cryptostegia. The analyses
showed that Iatex contains in addition to ruhher: proteins, phospholipids, amino
acids, glycerides, sugars, waxes, salts, quebrachitol, etc.
The composition of Hevea Iatex is different in the young tree vessels nearer
the cambium, from that in the older vessels as shown by ScHWEIZER, who believes
that tapping retards secondary growth of tree, a.nd that Iatex is formed at the
expense of the translocation current. Production of ruhher of 3-4 kgjtreefyear
may decrease the total secondary growth by 1/ 3 , whereas an intensive tapping
of 5 kg rubberftreefyear willlower the secondary growth to 1/ 2 • Simultaneously
 Chemistry of rubber. 227

with heavy tapping the symptoms of brown inner bark become apparent. It is
suggested that there is competition between growth and latex formation, since
during heavy tapping there is not present sufficient current of assimilates for the
formation of a quantity of rubber commensurate with the osmotic condition of
the cells. At such a time the phospholipids increase, the proteins decrease, accom-
panied by a fall in rub- Table 4. The values are in % of coagulated latex.
ber precursors. Latex
of very young growing Plant i Ash I Resins I Fats Protein IRubber
1

tissues contains very

small rubber particles Tabernaemontana heyneana/ 3.2 ~~1.4
118.21 2.7 I 5.2
Alstonia scholaris . . . . I 3.9 1.1 I 16.7 I 2.5 l 4.5 1
usually less than 0.5 mi- Oryptolepis buchanani . . 2.8 1.21 16.4 0.4 10.1
crons.
NAIR, SRIDHARAN and MADHAVAN studied the latex of various indigenous
plants of Travancore, and separated them into various fractions as shown in
table 4. The most important rubber producer was Cryptolepis.

Chemistry of ruhher.
Isoprene, a diolefin produced by the pyrrolysis of rubber and turpentine
has the following formula: CH 3
I
CH 2 =C-CH=CH 2
It is a C 5H 8 hydrocarbon which easily undergoes polymerization. Chemically
speaking isoprene may be considered to be the elementary building block of
terpenes and rubber. Gutta is a polyisoprene hydrocarbon produced among
other plants by Palaquium gutta and Mimusops balata. Rubber and gutta have
the same empirical formula (C5H 8 )x but differ in the spatial arrangement of
their carbon atoms about the double bonds, rubber being the cis, gutta the
trans isomer. This fundamental difference is Rhown below:
'I I
CH 2" ,c /CH 3 CHz"'c/CH3
II II
c c
CH/
I
""H H/ "'CH2
I
Cis-configuration about the double bond in Trans-eenfiguration about the double bond
the isoprene unit of rubber. in the isoprene unit of gutta.
Naturalrubber obtained by the normal coagulation contains 92-93% pure
rubber hydrocarbon. It has been established beyond doubt that rubber has a
polyisoprene structure, the linkages between the isoprene units are always 1-4
and also the junction is always head-to-tail; a case of 1-2 polymerization has
never been observed in natural rubber.
Rubber exists in nature only as the all-cis configuration whereas gutta is
the all-trans isomer. The stereochemical structure of rubber and gutta are then:
CH3, /H CH3, /H CH3, /H
'-C=C '-C=C '-C=C
-CH 2/ "'CH2-CH 2/ "'CH2-CH/ ""CH2-
Rubber hydrocarbon: Cis-eonfiguration

CH 3"'
/ CH2-CH 2" ' /H CH3"' /CH 2-
C=C, /C=C, /C=C
-CH 2/ '-H CH 3 '-CH2-CH 2 ""H
Gutta hydrocarbon: Trans-eenfiguration.
15*
228 BARBARIN ARREGUIN: Rubber and Iatex.

The chemical structure of both hydrocarbons have been established by ozonolysis

and by infrared absorption spectroscopy.
The degree of polymerization in a molecule of rubber is between 3,000 to
6,000 isoprene units. The molecular weight varies from 200,000 to 1,000,000
although fractions of smaller or greater molecular weight are often reported
in the literature. Microscopic investigation of Iatex particles from 16 species
of plants using the electron microscope was undertaken by HENDRICKS, WILDMAN
and McMURDIE, and the conclusions arrived at, indicated that some particleshave
a volume sufficiently !arge to contain only a few rubber molecules entwined
or packed into loose balls holding the other Iatex constituents between them.
Within the same Iatex particles of various sizes, coalescence was observed which
permitted the formation of pear shaped or rod shaped structures. The diameter
T bl 5 of the rubber particles observed ranged
a e · from 0.01 to 1.0 microns.
Type of
Plants hydrocarbon DE VRIES and DALITZ using, too, the
electron microscope techniques obtained
Hevea brasiliensis . rubber micrographs of Hevea Iatex gels which
Mimusopa balata . gutta showed an average rubber particle dia-
Asclepias erosa . . rubber meter of 0.28 microns without coales-
Solidaga leavenworthii rubber
Eucomia ulmoides . . . gutta cence. They indicated that coalescence
J atropha speciosa . . . rubber was prevented by the interfacial layers
Funtumia elastica . . . rubber of protein and soaps present in the Iatex.
Cryptostegia grandiflora rubber Chemical reactions are not good tools
Ficus elastica . . . . . rubber
Manihot dichatonia . . rubber in the identification of gutta and rubber.
Taraxacum kok saghyz rubber However their absorption spectra in the
infrared are different. Purified samples
of gutta from Mimusops balata Iatex and rubber from Hevea Iatex were studied
by HENDRICKS, WILDMAN and JoNES; the spectrographic data showed differences
in absorption in the region 11.8-12.3 microns. Thus at 12.0 microns the ab-
sorption coefficient of rubber is 42% greater than that of gutta, affording a
clear cut method for the differentiation of the two hydrocarbons. In connection
with this, it can be concluded that the naturally occurring elastomers can be
classified on the basis of the infrared data as belonging to the Hevea type or
the gutta type. SAUNDERS and SMITH arrived at the same conclusion. By this
method the polyisoprenic hydrocarbons from various plants have been divided
into the rubber and gutta classes as is shown in table 5. The molecular
weights of rubber and gutta obtained from plants in the last table showed a
wide range. For example the molecular weight of gutta from chicle as
determined by LEEPER and SoHLEBINGER was about 17,000 and exists in two
polymorphic forms.
In all the cases studied, any singleplant either contains rubber or gutta but
never both cis and trans hydrocarbons. This means that there is not inter or
intramolecular mixing. Thus a singleplant appears to contain the specificenzyme
necessary for the condensation of the units of the precursor exclusively in the
cis, or in the trans stereochemical configuration.
Using as a model isoprene itself it is possible that condensation may take
place through any end of the isoprene unit, and in this way an equilibrium
mixture is obtained for this synthetic polyisoprene, showing cross linking between
groups. Whereas polymerization in the plant takes place quite specifically,
either the cis or the trans configurations are formed throughout the entire hydro-
carbon molecule. It may well be that the same precursor is used in both poly-
 Chemistry of rubber. 229

merizations, biological and chemical, differing only in the specificity of the

enzymatic mechanism used by the plant to build up the cis or the trans isomer.
The rubber molecule is an extremely long chain molecule. If this molecule
were fully stretched it would have a length of about 2 microns. However the
rubber molecule is found coiled and entangled. Naturalrubber which is amorphous
at ordinary temperatures could be induced to crystallize by various agents:
Low temperatures which lower the thermal motions favour crystallization (tem-
peratures below 11° 0), forming crystallites within the rubber, thus one molecule
may have crystallized portions, the rest of it remaining amorphous. Crystalliza-
tion is likewise produced by pressure and by stretching; in the latter case the
molecules are stretched in the direction of the stress, acquiring a nearly parallel
arrangement. If it is plotted the percentage of crystalline rubber against elongation
it is found that crystallization is a function of the applied stress within certain
limits. Hence for a 500% elongation the crystallization is about 25%.
In gutta the steric factors together with the more stable trans form allow
it to exist in crystalline form. Gutta crystals obtained from chicle by ScHLE-
SINGER and LEEPER were optically anisotropic and were 0.35 mm long.
X-ray analysis of rubber indicates a repeating unit cell of about 8.1 A along
the chain axis, which will correspond to two isoprene residues. In gutta the
identity period had a dimension of 4.8 A indicating only one isoprene in the
unit cell.
The isomeric hydrocarbons gutta and rubber are obtained commercially
from a few plants, in these plants they occur as traces or as major constituents.
Commercially only those plants with a high rubber content are used, such as
Hevea and to certain extent the Russian dandelion and guayule.
Rubber analysis. The methods of analysis currently make use of the reactions of the
double bonds, particularly addition reactions such as the addit.ion of bromine. This addition
reaction has proved tobe the most reliable method for the analyl'is of ruhher hydrocarbon.
In crude ruhher other than Hevea rubber, where the non-rubber components are not
very weil known, or are present as bulky undesirable contaminants as in the case of
guayule, the Russian dandelion and Cryptostegia, it is convenient to submit the ruhher to
purification. Rubber is easily soluble in benzene, petroleuro ether, ether, carbon disulfide,
chloroform etc., but it is insoluble in acetone and methanol. Thus solvent purification of
ruhher is quite important before analysis.
The methods of ruhher analysis can be classified as gravimetric and volumetric. Some-
times in the gravimetric procedures the purified ruhher is weighed out as such, but more
often the bromide of ruhher is made and determined quantitatively by gravimetry. This
method is applicable only to unvulcanized rubber. Another method using a colorimeter
consists in the precipitation of the ruhher from a ruhher solution by the addition of methanol
and its determination by turbidimetry, see WmTTENBERGER and BRICE. The ruhher bromide
and the Oxidation with chromic acid are examples of volumetric methods. A good account
of these procedures is found in the article by WILLITS, SWAIN and Ooo. BEKKEDAHL in
various reviews has discussed the methods of ruhher analysis including the chromic acid
oxidation, however the most widely used procedure is the gravimetric determination as
bromide which is reliable when the impurities capable of reacting with bromine are absent.
The additionderivative between bromine and ruhher was prepared originally by GLADSTONE
and HIBBERT. Later it was shown that the so-called tetrabromide derivative was insoluble
in alcohol, a solvent which indeed became very useful for precipitation. The addition com-
pound as shown earlier by WEBER had the following empirical formula: C10H 16Br4 •
The ruhher bromide method was used by EDISON (see BoNNER and GALSTON) in a very
extensive survey for ruhher in several hundred native American plants.
In general the first steps for ruhher analysis consist of extracting the Iatex by tapping
or by crushing and coagulation which are really mechanical operations. · Coagulation of
Iatex by heat is followed by solvent extraction in order to purify the hydrocarbon. The
more common solvents in tbis step are: water, ethanol and acetone which dissolve non-
ruhher impurities. The ruhher is then dissolved in benzene and treated with bromine water
or bromine in carbon tetrachloride. Other common solvents for ruhher used frequently
are carbon tetrachloride, chloroform etc. The bromination is effected by allowing the bromine
230 BARBARIN ARREGUIN: Rubber and Iatex.

to remain in contact with the rubber solution for a certain length cf time, generally at low
temperature to prevent brornine substitution reactions. The rubber brornide forrned is
precipitated by adding ethyl alcohol, filtered and dried in a vacuurn oven. The weight of
the rubber bromide is converted to rubber hydrocarbon by multiplying by a conversion
factor.
If we suppose that the bromine adds completely to the double bonds a rubber bromide
of the following specific composition should be formed: C5H 8 Br 2 , and the theoretical con-
version factor is 0.298. A factor of 0.301 was obtained fcr guayule rubber and of 0.299 for
Hevea rubber. SrENCE and CALDWELL did a great deal of the preliminary analytical work.
In an improved method, GowANS and CLARK studied the effect of various factors such as
light, temperature, concentration of bromirre and rubber on the formation of the rubber
bromide. Recently a fast and accurate method of the preparation of samples for rubber
analysis in the guayule was reported by MEEKS, CROOK, PARDO and CLARK who claimed
that it ruptures completely the cells of guayule plants permitting a complete rubber recovery.
In the volumetric analysis of the rubber, an excess but known amount of bromine in
carbon tetrachloride is added to a rubber solution and after a few hours potassium iodide
is added, the latter willliberate an equivalent amount of iodine to the excess bromine, then
it can be titrated with standard thi03ulfate solution. By adding potassium iodate the sub-
stituted bromirre is found. By difference in the thiosulfate titrations the bromine added
to the double bonds is measured. F ANNING and BEKKEDAHL reported the use of the refractive
index measurements in the quantitative determination of rubber using 1-bromnaphtalene
as solvent.
Crude natural rubber contains in addition to rubber hydrocarbon: resins, proteins,
ash, and other substances. The chemical and physical analysis of rubber from various sources
indicate that they only differ in the degree of polymerization and in the contaminants present
in the crude material. At the present the most widely used raw material by the rubber
industry is the smoked sheet from Hevea, all other rubber bearing plants play a secondary
role. Nonetheless it can be said that if rubber from some secondary sources is purified free
from undesirable contaminants, it would prove to have the same tensile strength and abrasion
resistance as Hevearubber. A good example of this statement is made for the rubber of golden-
rod by :M:cKENNON, n'AQurN, GurLBEAU, MoLAISON, PoLMINSKI and Vrx.
New methods of determination of rubber and gutta in plants are compiled by BENEDICT.
Molecular wcight of rubber. Highpolymersare usually mixtures of molecules
having different degrees of polymerization, and this is also true for natural rubber.
The molecular weight of rubber can be determined by osmotic pressure and
viscosity measurements. A difficulty with the osmotic pressure procedure is the
preparation of a satisfactory membrane, thus this method cannot be used for
the investigation of solutions containing molecules of molecular weight less than
10,000. Likewise polymers with molecular weights above 500,000 require extra-
polation. The common procedure consists of measuring the osmotic pressure
of solutions of rubber in benzene, chlorobenzene, toluene, or in mixed solvents
such as benzene containing 15% methanol, which was used successfully by GEE.
The viscosity measurements of liquids and solutions containing chain like
molecules, afford valuable information on the average size of the molecules
of the substance under investigation. What one measures by viscosity is the
average value of the actual molecular weight distribution curve. STAUDINGER
proposed the first mathematical formulation of the relation between the viscosity
of a solution and the molecular weight of the solute. In this method solutions
containing less than 0.1% rubber are used.
BLOOMFIELD (1, 2, 3) summarizes the results of four years of investigation
with Hevea, which indicated that the molecular weight of rubber determined
in freshly tapped Hevea latex by the viscosity and osmotic procedures ranges
from several millions to less than one hundred thousands, with an approximate
average of one million. The major part of the Hevearubber lies in the upper range.
The resolution of rubber into fractions of various molecular weights has
been attempted by MmGLEY, HENNE, and RENOLL. Through acetone and
alcohol precipitation of the dissolved rubber VERGHESE, and also BLOOMFIELD,
have tried to fractionate the rubber by stepwise addition of acetone or methanol,
 Notes on the potential rubber bearing plants. 231

and separation of the fractions produced. If air is practically excluded in the

entire process from the initial collection of ruhher, it is found that only a small
proportion of the ruhher is present as low molecular weight fraction. BENEDICT,
BROOKS and PucKETT studied the molecular weight of ruhher from various
plant parts of the guayule. Their data show that the roots contained the highest
molecular weight ruhher, followed hy the stem, next the hranches and finally
the tips. The highest molecular weight determined hy viscosity was 210,000,
and the lowest 20,000.

Notes on the potential ruhher bearing plants.

Systematic studies for ruhher analysis have heen made on desert plants hy
BucHRER and BENSON. The plants analysed helonged to the following families:
Euphorbiaceae, Asclepiadaceae and Apocynaceae. In other countries, too, surveys
of ruhher hearing plants have heen given, and the possihility of growing such
plants has heen studied hy NAIR, SRIDHARAN and MADHAVAN; the latexes of
Alstonia scholaris, Tabernaemontana heyneana and Cryptolepis buchanani, all three
helonging to the Travancore minor forests wue investigated.
Many of the plant families of Asia, America, and Africa produce ruhher.
The most important are: Euphorbiaceae, A pocynaceae, Urticaceae, Asclepiadaceae,
Moraceae, Compositae, Sapotaceae, Musaceae, Convolvulaceae, Menispermaceae,
Loranthaceae, Celastraceae etc. Hevea brasiliensis which is an Euphorbiaceae is
no douht the one preferred hy the planters, although there are various species
of Hevea. Other ruhher containing plants are Jelutong in Malasia, Ficus elastica,
Taraxacum kok saghyz in the steppes of Russia, the Ceara or Manihot glaziovü
in South America, Castilloa elastica, Parthenium argentatum in Mexico, and also
some ruhher hearing lianas in Africa, etc.
Hevea. Hevea brasiliensis is the most important producer of natural ruhher
in the world. This tree is native to the Brasilian jungles in South America.
During many years this part of the world was the solenatural ruhher supplier.
Later the first seedlings were raised from seeds in London at Kew Botanical
Gardens, and these were sent to Asia for their propagation and cultivation. Since
then this tree has been submitted to very careful studies designed mainly to
improve ruhher quality and yield. A very good account on the status of Hevea
in the Far East is found in the hook hy DIJKMAN. BRANDES, too, presents the
conditions of Hevea cultivation in the Western Heinisphere.
By individual selection, pollination, clonal propagation and budding with
high yielding strains, it has heen possible to obtain as much or even more
than 2,000 lhs. of ruhher per acre per year. The trees are tapped when they are
at least five years of age. The latex vessels in the Hevea are produced by
the cambium in successive rings, and they extend longitudinally throughout
the trunk of the tree. In radial direction they show very little brauehing and
intercommunication with inner or outer vessels. The vessels of any single ring
intercommunicate well. This anastomosis of the vessels forms the reservoir
from which the latex comes out after tapping. The tapping goes on throughout
180 days of the year, and begins near the level of the ground. The tapping
period is followed hy a rest period, the latter is quite important for the recovery
of the tree and assures the highest yields.
The latex flows out of the cut tap and it is collected in special reservoirs.
The ruhher present in the flow of latex from a tap diminishes with time, however
it has been noted that this decrease is accompanied by an increase in the volume
of latex, resulting in a net increase of ruhher ohtained in the first few days.
232 BARBARIN ARREGUfN: Rubber and Iatex.

This phenomenon is called the dilution reaction. The osmotic pressure of latex
is about ten atmospheres, and once the cut is made the osmotic balance with
adjacent cells is disturbed causing water to be taken by the latex from neigh-
bouring cells, bringing about this dilution. Newly formed rubber will enter the
latex flow showing that the mechanism for rubber formation is still active,
and has not been destroyed or drained away. Hevea trees 8 to 14 years old
produce about 10 kg of rubber per tree per year.
Russian dandelion. A Russian survey for rubber bearing plants showed that
the Russian dandelion or Taraxacum kok saghyz is the most promising rubber
producer in the higher latitudes. Originally kok saghyz was found in Central
Asia by an expedition headed by VAVILOV, and it was discovered by RoDIN,
a member of the expedition, on the high plateau of the Tien Shan mountains
near Mongolia. WHALEY and BowEN published a comprehensive paper on
Taraxacum kok saghyz. Studies on the anatomy and morphology of kok saghyz
were carried out by ARTSCHWAGER and McGumE.
Taraxacum kok saghyz is a herbaceous perennial Composita which produces
the rubber mainly in the roots. The plants have a tap root with some brauehing
within which the latex vessels formed in the secondary phloem increase in
number and size in the second year of development. The plant reproduces
by seeds planted usually before the winter, and the flowering continues until
the fall. The best suited soils for the growth of this plant are the black soils
of Chernozem type, with abundant water.
The rubber is laid down in the connected system of the lactiferous vessels
and increases during the first two years followed by a subsequent decrease.
For this reason the plants are harvested at the end of the first year of growth
or in the spring of the second year. The extraction of rubber from kok saghyz
is similar to the method employed for guayule, and consists of treating the
harvested root with hot water to leach out soluble substances and to soften the
tissue. The rubber coagulates in filaments and by milling the roots in the presence
of water, the rubber comes out and it is collected on a vibrating screen. By
repeated flotation and vibration all the rubber is finally obtained. The yields
of rubber from kok saghyz are variable, ranging from 4 to 5% on dry root basis.
Figures of about 100 lbs of rubber per acre per year were obtained in the United
States. However values of 400 lbs per acre per year have been reported.
Tau saghyz is also a rubber containing plant that surpasses kok saghyz in
hydrocarbon content. However its smaller seed production, and difficult propaga-
tion make it less economical. Krym saghyz or Taraxacum megalorrhizon shows
good rubber yields but the plants do not survive well the winter.
Guayule. Parthenium argentatum Gray has its native habitat in the desert
areas lying between Mexico and the United States at elevations from four to
seven thousand feet. These areas receive from seven to fourteen inches of rainfalL
Guayule is a shrub extremely resistant to hot and cold temperatures. In articles
by LLOYD, EsAu, ARTSCHWAGER, and by TAYLOR the history, anatomy and
potentiality as rubber producer is extensively discussed. The data indicate that
in the guayule, rubber is present in all organs of the plant. The commercially
important portions are the bark of the roots and stems, where rubber is deposited
in the parenchymatous cells of the phloem and cortex. The content of rubber
in guayule is proportional to the bark and wood ratio.
The guayule does not possess lactiferous vessels, in this plant the rubber
is found as particles suspended in the latex present in the parenchyma cells.
Guayule is a very active accumulator of rubber, and concentrations up to 16%
or even more based on the dry weight basis have been frequently recorded in
 Environmental factors in the biosynthesis of rubber. 233

old plants. The plant reproduces by seeds and shows apomixis. Plantings of
guayule directly from seeds have yielded between 1,200 to 1,500 lbs of rubber
per acre in less than two years of growth. An average figure of 400 lbs rubber
per acre per year under normal growing conditions is easily obtained. The growth
period from seed to harvest time may be two to seven years or possibly longer,
since guayule continues to manufacture rubber as it ages. Irrigation and proper
spacing according to TrNGEY ·will induce yields up to 1,700 lbs rubber per acre
in 33 months.
Guayule rubber softens and even liquefies showing an apparent inferiority
as compared with Hevea plantation rubber. By eliminating the resins present
in the guayule crude rubber either by microbial action as described by ALLEN,
NAGHSKI and HooVER; and by WHITE, NAGHSKI, ALLEN, HoovER and WrLLA-
MAN, or by the use of acetic acid extractions as reported by NrsHIMURA, HrRO-
SAWA and EMERSON a good quality rubber is obtained which compares favorably
in tensile strength with Hevea smoked sheet.
Other species of Parthenium such as incanum are much less important since
they produce very little rubber, whereas the essential oil output is quite large.
Cryptostegia. Cryptostegia grandiflora is an Asclepiadaceae indigenous in
Africa, now growing in the tropical and subtropical America. It possesses long
stems, whip like and almost leafless, where the rubber is accumulated in the latex
vessels and parenchyma cells. Up to two percent rubber has been detected in
the leaves, but the greatest proportion is present in the stems. The anatomy
and latex system is discussed in articles byBLASER; ARTSCHWAGER and BoNNER
and GALSTON too. The process of getting the rubber consists of tapping the
whips of Cryptostegia, after which the rubber is coagulated and purified. The
leaf rubber is a very low molecular weight polymer and consequently of not
very good quality. The rubber yields are about 200 lbsjacre.
Goldenrod. Rubber has been extracted from goldenrod. It contains rubber
in the leaves, and yields up to 6.3% on the dry weight basis. This plant has
considerable historical importance since it was considered by EmsoN and others
as an economical rubber plant which could be cultivated in temperate zones.
The method of extraction consists of a preliminary acetone extraction of the
dried leaves in order to remove resins. Then by extraction with benzene, and
by acetone precipitation a tough raw rubberwas obtained that was easy to handle.
The best species are Solidaga sempervirens, S. leavenworthii, and S. mexicana,
all three can be cultivated. The agricultural yie1ds of goldenrod are between
225 and 450 lbs. rubberjacrejyear.

Environmental factors in the hiosynthesis of ruhher.

~Iineral nutrition and fertilizers. Since each species of rubber bearing plant
has its own nutritional requirements, it is rather difficult to integrate and sum-
marize the data existent on these matters for all rubber containing plants. Techni-
ques have been designed for nutrition experiments of Hevea, and mineral analysis
of the leaves and latex by BEAUFILS, with special emphasis on Ca, K, Mg, N,
P, Cu, Mn, and Fe. The result of the analysis indicated that there is a great
deal of variation in the ash content and its composition. As has been pointed
out by CHAPMAN it is necessary to carry out many determinations in order
to obtain a good statistical analysis. He found that leaf mineral composition
and growth rate of Hevea seedlings showed a correlation. In mature rubber
plants the same direct correlation exists; also latex yield was correlated es-
pecially with leaf nitrogen.
234 BARBARIN ARREGUIN: Rubber and latex.

In experiments using fertilizers containing P, N, K, NP, PK, NK, and NPK

and controls, it has been found that for Hevea the fertilizers containing K, induced
the greatest rubber production, and that the K content of the leaves of the
same trees undergoes changes throughout the year, with higher content shortly
after the application of the K fertilizer. Only where a K containing fertilizer
had been given, were significant increases of K found in the leaves. A similar
but minor effect was detected in the case of P., and Mg. The four elements K,
Ca, Mg, and Mn, show a close interrelationship. The first element antagonizes
with the other three, the correlation coefficients also indicate that the K fluctua-
tion in the leaves follows in mature plants the same fluctuations as copper. In
general it can be said that K is the most important element in Hevea culture.
TIXIER and BEAUFILS showed that plants given P have the best growth.
There is, too, a beneficial effect of K on rubber quality, which seems to indicate
an important and complex role of K on latex physiology. The leaf analysis
was done on the dried powdered leaves, or on the fresh latex. A marked retarda-
tion of growth was detected by BaLLE-JaNES in Hevea seedlings with restricted
supplies of the following major nutrients: N, S, P, Mg, Ca, Fe, Mn, and visual
deficiencies became apparent except in the case of Ca, and B. The greater the
mineral supply, the greater the amount of rubber in the stems and petioles of
Hevea. This effect changed as the age of the seedlings progressed. It can be
concluded that Hevea requirements for Ca, and B are very low.
InkoksaghyzBARANaVSKii reported that the addition of PN fertilizers produced
latex with more dry solids, bringing about increases in rubber content, and
also in the molecular weight of it. After the addition of N and P, the maximum
molecular weight reached 184,000 at the fruiting stage.
Likewise according to KALINKEVICH, N given as ammonium sulphate will
cause a faster maturation and aging of the kok saghyz plant, than nitrate N.
Then plants treated with nitrate N, showed a slower development and were
physiologically younger. In general plants grow better with a nitrate diet, than
with ammonium salts. Furthermore during formation of the latex vessels KALIN-
KEVICH suggests that the best diet is nitrate, but during rubber deposition am-
monium salts treatment is the indicated one.
LJUKaVA studying the effect of mineral fertilizers on experimental plantings
of kok saghyz, particularly those containing superphosphate alone or Super-
phosphate-ammonium nitrate, up to 1: 1 ratio of P and N, showed that plants
receiving P fertilizers gave higher content of rubber in the latex, and a beneficial
effect was observed over the controls with the addition of the PN fertilizer.
A linear function is found between the size of latex globules and the degree
of polymerization of rubber contained therein. The quality of rubber is improved
with increase in latex globule size. The use of PN fertilizers also stimulates
the development of the latex channels of the plant.
In guayule, MITCHELL found that growth and rubber accumulation is propor-
tional to the N and P supply. Ca, K and Mg, had little effect. In greenhause
experiments, guayule plants grown in gravel culture over a period af eight months,
BaNNER showed that growth and rubber accumulation was increased in plants
supplied with N, particularly in the form of nitrate. The accumulation of rubber
was rather independent of the concentration of Ca, and K.
Temperature. Temperature has a definite effect on the rubber productian
by guayule plants. In greenhause experiments with potted plants, BaNNER
found that if night temperature was kept low (between 40-45° F.}, during 16 hours,
the rubber produced was substantially increased. A constant temperature either
low or high during the 24 hours was less effective. Thus it seems that low night
 Enzymes present in the latex. 235

temperatures in guayule causes an induction of rubber production, and such

cold temperatures are common in winter in the desert areas situated in Northern
Mexico. The guayule plants grown at constant air temperature, but variable
soil temperature behave similarly, since BENEDICT found that the rubber and
resins decreased as the soil temperature increased from 4-18° C.; he concludes
that low soil temperature will tend to increase the rubber percent in guayule
plants, but the high soil temperature will not necessarily maen low rubber content.
For kok saghyz the temperature requirements are not so narrow at least
with respect to growth, since it has been reported that this plant has been
cultivated as far north as Archangel. It is no doubt a cold climate plant whereas
Hevea is a typical tropical plant.
Plant hormones. Some reports on the effect of plant hormones on the produc-
tion of rubber have appeared, particularly in Hevea. The results of CHAPMAN
suggest that local application of plant hormones below the tap in Hevea trees
increased the latex yields. The application above the tapped region decreased
the yield. It is rather difficult to reconcile at the present these opposite effects
in a simple explanation. It is suggested that the increased latex production
is due to Ionger flow periods.
Effect of copper salts. During the last few years the results obtained with
injections or application of copper salts specially in the form of sulfate have
been published. These experiments in Hevea trees were dorre in the following
manner: two holes of about 1 cm in diameter and 4 cm deep are drilled in the
trunk of the tree, and the copper salt preferably as a powder or tablet is placed
in the holes. The results obtained by CoMPAGNON and TIXIER; and TIXIER,
showed that the best increments in rubber or latex were obtained when the
applications took place at the top of the tapping notch. The salts which induced
increases in rubber formation were CuS0 4 and to lesser extent MnS0 4 ; other
salts like ZnS0 4 caused decreased rubber production. NaF caused an initial
increase followed by a considerable decrease. CuS0 4 passes rapidly into the latex
flow. The response to its application is not yet well understood, and in the
case of CuS0 4 this response may last from 3 to 6 months. Increases up to 40%
or in some cases even more were produced as compared with the untreated
controls. It seems that the best time for the injections is at the end of the dry
season. The increased rubber yields from treated trees are not at the expense
of future productivity, inasmuch as the tree continues to make more rubber
than the controls several months after the treatment.
These workers, too, in further experiments coated the surface of the tapping
panel previously scrapped but not to the point of latex flow, with sodium
2,4-dichlorophenoxyacetate. The application of this salt raised the formation
of latex by 40 to 100% but with a lower concentration of rubber.
Grafting. ZHDANOVA reported in experiments with Rudbeckia bicolor grafts
on kok saghyz, that during the active growing the kok saghyz portion produced
large amounts of rubber; this increase was detected not only in the older parts
of the plant, but also in the newly formed rootlets. These results suggested
that rubber synthesis took place at the expense of a photosynthetic product
formed in the Rudbeckia leaves, which had moved down through the graft.

Enzymes present in the Iatex.

The proteins in the latex are partly adsorbed on the surface of the particles
suspended therein, the rest existing free in the serum. The proteins from Hevea
latex serum were studied electrophoretically by RoE and EwART, using samples
236 BARBARiN ARREGUiN: Rubber and Iatex.

of serum obtained from fresh latex of Sumatra and Florida. They found that
the serum could be resolved into seven distinct components at PH 6.85 and
PH 8.43. Both serums were similar since the nurober of components was the same,
and the relative mobilities and abundance of fractions were very much alike
However it was noticed that Florida serums had sharper peaks which indicated
a greater electrophoretic homogeneity. If ammonia was added to the latex
prior to the removal of the serum, changes in the electrophoretic mobility of
the proteins were detected and the proteins could be resolved into two com-
ponents only.
Some proteins in the latex possess enzymatic activity. Thus ÜHEAH found
in fresh Hevea latex a phosphatase which hydrolyzes glycerophosphate from PH 5
to 7. This enzyme was obtained by coagulating the latex with acetic acid. The
activity remained in the serum, the enzyme then could be purified further
by ammonium sulfate precipitations.
· Very strong peroxidase activity was shown by DoMAN in the latex of Eucomia
and Evonymus. This activity was of the order of magnitudeofthat for fig and
horse raddish plants which are noted by their potent peroxidasic activity.
Evonymus a plant which stores most of its rubber in the root has the highest
peroxidase activity in the root, whereas Eucomia which distributes its latex
throughout the plant has a greater uniformity in the distribution of the enzyme.
Ml:KHLIN and BRONOVITSKAYA determined the peroxidase activity in kok
saghyz roots and concluded that catalase and peroxidase activities decline as
rubber is synthesized or as the molecular weight of the rubber increases in the
root, while polyphenol oxidase activity remains constant. In the latex of kok
saghyz, peroxidase was absent and catalase was present only in small amounts.
It was shown, too, that latex reduces methylene blue, but this ability is destroyed
by boiling. The reduction of methylene blue was accelerated by theaddition
of succinate, glutamate, fumarate and malate ions, as well as by small amounts
of ethanol and glyceraldehyde. Citric acid was ineffective. Other enzymes which
were determined in the latex were alcohol dehydrogenase and malic and glutamic
acid dehydrogenases.
HAAN-HOMANS studying Hevealatex or fractions of it identified the following
enzymes: catalase, tyrosinase, peroxidase and some inhibitory substances. After
the preliminary experiments of HAAN -ROMANS, ÜRETIN measured the oxidation-
reduction changes of latex using methylene blue as indicator. It was found
that the PRof the latex changes from 6.9 to 6.2 after the first few hours of tapping.
Addition of KCN, NaN3 or NaF or other inhibitors of enzymes containing Fe
or Cu as prosthetic groups, did not change the character of the oxidation-reduction
potential, andin general reagents which precipitate heavy metals did not change
it. It was suggested that the negative potential of latex may be related to the
presence of the thiol functional group.
MIKHLIN and PsHENOVA stated that most of the polyphenol oxidase activity
of the kok saghyz root existed in the latex and some polyphenolic compounds
which probably acted as natural enzyme substrates were isolated. Furthermore
proteolytic activity has been often ascribed to latex derived from various sources.

Evaluation of the theories on ruhher hiosynthesis.

Once the chemical structure of a natural substance is established it remains
to answer the question of how this substance is synthesized in nature and to
determine the sequence of reactions involved in its biosynthesis. One of the
first and essential steps is to know the substance or substances from which
 Evaluation of the theories on rubber biosynthesis. 237

the ultimate product is derived, this would require to know the chemistry from
the initial precursor up to the terminal compound, and the biological systems
by which the plants are able to carry out such specific synthesis which in our
particular case is the synthesis of the complex polymer termed rubber.
In the present work the study of the biosynthesis of polyisoprenes will be
restricted exclusively to rubber. It is however true that no investigations have
been made on the biosynthesis of gutta. In spite of this it may well be that
all the steps involved in the synthesis of rubber are the same for the synthesis
of gutta, with the exception of the polymerization: plants containing gutta quite
specifically form the all-trans hydrocarbon, whereas those containing rubber
will make only the all-cis isomer or rubber.
In connection with the biological synthesis of rubber it is worth mentioning
here, that considering isoprene as the building block, the isoprene rule has been
brilliantly postulated by RuziCKA based on chemical grounds. He states that
the carbon skeleton of terpenic compounds is composed of isoprene units linked
in a regular or irregular arrangement. This empirical rule was deduced from
the structure of the natural terpenes. In the following scheme the formation
of a non-cyclic substance through the junction of two isoprene units is shown.
0 0
I I
0-0-0-0-0-0-0-0
Regular arrangement: head-to-tail.
0 0
I I
0-0-0-0-0-C-0-0
One possibility of irregular arrangement: tail-to-tail.
0 c
I I
C-0-C-C-C-C-C-0
A second possibility of irregular arrangement: head-to-head.
The isoprene rule holds well for sesqui-, di- and triterpenes with some variations.
However as a biosynthetic path for rubber, this theory has the drawback that
in all rubber bearing plants the isoprene or building block has never been isolated.
The experimental results obtained in some cases indicate that essential oil
synthesis in plants may be antagonistic to the formation of rubber by the same
plant. Typical results of this kind were gained by PROKOFIEV (see BoNNERand
GALSTON). Allhis data indicated that as rubber formation increased essential oil
synthesis decreased. Identical results have been obtained in successive seasonal
determinations of rubber by SPENCE and McCALLUM for the desert shrub
guayule, which produces rubber preferentially in late fall and winter, at the time
where essential oil formation is at a minimum. By cutting the flowers as they
become apparent it is also possible to increase rubber accumulation.
In another case WrLDMAN, ABEGG, ELDER and HENDRICKS observed that
the latex of Cryptostegia madagascariensis contains as a major constituent a triter-
pene ester of the lupeol type, and only very minute amounts of rubber, whereas
the opposite is found in C. grandiflora. In crosses between them, the investi-
gations of the progeny suggested that rubber and the triterpene have a common
precursor which by non-cyclic condensation produces rubber or triterpene when
cyclized. PoLHAMUS basing his ideas on the crosses between the same two plants
suggested that essential oil formation is inherited as a recessive Mendelian
character, and that rubber formation is inherited as a dominant. The possibility
238 BARBARIN ARREGUIN: Rubber and latex.

here exists that both isoprenoids are polymerized from a common precursor,
which in the presence of a specific enzyme genetically controlled (see BEADLE
and TATUM), is converted either to rubber or essential oils. Kok saghyz, Castilloa,
Ficus, Chicle, and many Asclepias and Euphorbia sp. etc. contain triterpenes
in addition to rubber or gutta. These triterpenes which are closely related to
rubber have been carefully reviewed recently by LEMIN and SANDOVAL.
The biogenesis of rubber was postulated by RARRIES (see MEYER) to occur
via levulinic aldehyde. The latter substance which is obtained in considerable
quantities by the action of ozone on rubber plus subsequent treatment with
water, has the structure shown below:

?H 3

0=C-CH 2-CH 2-CHO

a b
Then it is suggested that structure a) is converted to b).

The biogenesis of the isoprene molecule in a review by PoLONOVSKI has

been suggested to occur through an aldolization reaction between acetone and
acetaldehyde, followed by the loss of two molecules of water. These reactions
are presented in the next scheme.

-2H,O
-----------? ----?

Acetaldehyde occurs in the latex of Hevea and acetone has been detected in
the leaves, flowers and fruits of the same tree. The latter proposal suggests
that the precursors of isoprene are smaller molecules, which condense to yield
a C5 compound which may be transformed into isoprene. Among the possible C5
substances which could be used in isoprene synthesis we may cite here: leucine,
amyl alcohol, valeric acid, all three of common occurrence in plants.
The diene synthesis is based on the direct union of a diene with another
unsaturated system called the dienophil, which generally contains a double or
triple bond. The two substances condense forming a cyclic structure. These
reactions often happen spontaneously. A condensation of dienes through a
different mechanism could possible lead to an acyclic structure e.g.:

However this union would not produce a cis diisoprene, and therefore it may be
concluded that the diene synthesis has no practical application in the synthesis
of the building block for rubber.
The basic similarity in the structures of substances as carotenoids, squalene,
sterols, triterpenes, vitamins A and D etc. and their resemblance to isoprene,
suggests that the biosynthesis of all of them could occur from isoprene as
starting material. Biologically this assumption has very little support. The
fact that isoprene in a free state is not found naturally does not necessarily
preclude it from being a precursor. Since isoprene is a reactive substance and
is easily converted to polymers, this reactivity could account for its absence
in a free state. In spite of it, many theories are based on the polymerization
of C5 compounds, a chain length which would favour their conversion to rubber.
 Evaluation of the theories on rubber biosynthesis. 239

In the review by BoNNER and GALSTON a summary table is given which

shows the chemical reactions having some connection with the formation of
Table 6.

H
CH3"' I_
1 ----+ /C-CH 2-C- 0
CH3/ I
OH
acetone acetaidehyde ß- h ydroxyisovaleraldeh yde

H H
CH3" I -H,O CH 3 , I
2 /C-CH 2-C=0 ----+ '-C=CH--C=O
CH3 I CH 3 ,f
OH
ß- hydroxyisov aleraldehyde ß-methylcrotonaldehyde

H
CH3"' - I_
/C--CH-C-0
+2H
CH 3 -H,o
ß-methylcrotonaldehyde isoprene

4 I

acetone acetaldehyde 4-hydroxy-2-pentanone

H
CH3, I
5 2 '-C=CH-C=O
eH/
3

ß-methylcrotonaldehyde citral

OH 0
CH3, I ii
6 - )C-CH 2-C-COOH
CH3
acetone pyruvic acid y- hydroxy -()(-ketoisoca proic aci d

y-hydroxy-a-ketosiocaproic acid ß-hydroxyisovaleraldehyde

a-ketoisocaproic acid isovaleraldehyde

a C5 suhstance that could give rise to ruh her. These data are presented in table 6.
As working hypothesis some of these reactions have been tried experimentally.
Another view indicates that ruhher is synthesized from pentoses (see AMBROs)
through pentadienil as an intermediate, which immediately condenses with itself
240 BARBARIN ARREGUfN: Rubber and latex.

to form rubber. The only support for this idea is that very small amounts of
levulinic aldehyde are recovered from the decomposition of ozonized rubber.
From certain experimental information it was concluded that serum latex contains
polymerization enzymes capable of converting isoprene to rubber. It was indicated
that oxygen was required for the reaction and that HCN acted as a poison.
Other workers like PRoKOFIEV proposed that isoprene is an intermediate
in the synthesis of not saturated hydrocarbons, and that this isoprene arises
from the condensation of acetone and acetaldehyde, forming ß-hydroxyiso-
valeraldehyde, which then by dehydration goes over to ß-methylcrotonaldehyde
which is finally reduced to isoprene. In support to this proposal it can be mention-
ed that there are some enzymes in latex which help in the polymerization of
butadiene to rubber like materials.
The hypothesis of EuLER that the formation of branched chains, as poly-
isoprene occurs by the condensation of acetone and acetaldehyde was tested
by KuziN and NEVRAJEVA who carried out this condensation under laboratory
conditions and in the presence of acid, base, or KCN; the condensation always
led to an unbranched product which was identified as 4-hydroxy-2-pentanone;
this reaction is indicated in the next scheme:
0 0 0 OH
II II II I
CH3-C-CH3 + CH-CH 3 CH 3-C-CH 2-CH-CH3
acetone acetaldehyde 4-hydroxy -2-pentanone

However using as a catalyst glycine impregnated cotton in a water free medium,

this condensation took place through the enol form of acetone as indicated
below:
H OH H
CH3"" I CHa I I
f'C-OH + CH -C=0
3 )c-CH 2-C=0
CH 2 CH3
acetone (enol), acetaldehyde ß-hydroxyisovaleraldehyde

Thus the condensation to ß-hydroxyisovaleraldehyde occurs in vitro; this con-

densation has not been proved in vivo, or under the influence of enzymes. Neither
4-hydroxy-2-pentanone nor ß-hydroxyisovaleraldehyde have been found in plants,
consequently this hypothesis has little biological implication.
The possibility that citral may act as a rubber precursor is left open to proof.
KREMERS studied the biogenesis of peppermint, and considered citral as forming
part of it.
Stillother workers consider carbohydrates as the raw material for the synthesis
of rubber, here may be cited NEIMAN, PROKOFIEV and SHANTAROVIOH who
studied kok saghyz. The leaves of this plant were painted with a 1% solution
of 0 14 labeled sucrose prepared biologically, and containing a small amount
of labeled glucose. The roots of the treated plants were suspended in a moist
chamber and tapped daily to get the latex. Radioactive carbon appeared in
the latex within the first ten days of the experiment, and its presence in rubber
proper was ascertained by the isolation of the purified ruhher and the determina-
tion of its 0 14 content. No 0 14 was determined in the water soluble components,
but 0 14 was present in substantial amounts in the acetone soluble components.
The conclusion was made that some products of sugar degradation or trans-
formation are the precursors of rubber. It is quite likely that resins were present
among the acetone soluble substances.
 Evaluation of the theories on ruhher hiosynthesis. 241

DROBKOV advocates the following scheme of reactions for the biosynthesis

of rubber:
Inulin --+ monosaccharides --+ acetone and acetaldehyde --+ isoprene --+ ruhher
This proposal has as initial intermediates the sugars.
Recently ALTMAN proposed a new theory for Hevea, in here the rubber is
synthesized in the green parts of the plant directly from carbon dioxide. The
rubber is formed first as a low molecular weight polymer, probably a liquid
which is translocated as a highly dispersed oil in water emulsion, in which the
globules of rubber are protected by a layer of a complex phospholipid. This
low molecular weight polymer is deposited as a solid anywhere in the plant,
the polymerization probably taking place in the Iatex vessels. The formation
of incrusting materials built by c5 molecules such as pentosans, hemicelluloses etc.,
according to this view may come from the oxidative breakdown of rubber.
An experimental survey of substances related structurally to the carbon
skeleton of isoprene was undertaken by BaNNER and ARREGUIN. The aim of
the investigation was to elucidate their potentiality as rubber precursors in
guayule seedlings as well as in older plants. The test substances were added
to the nutrient solutions. The increments of rubber over and above the controls
were considered to be a measure of the utilization of such substances in rubber
synthesis. Of the substances tried those which induced increases of rubber were:
glycerol, acetate, acetoacetate, acetone, and ß-methyl crotonic acid. The latter
substance could arise from the condensation of acetic acid and acetone in the
following way:

acetone acetic acid ß-methyl crotonic acid

In support to this reaction it may be mentioned that acetone has been found
in guayule.
Studies with isolated stem sections of guayule by ARREGUIN and BaNNER,
in which callus formation was induced by the addition of indoleacetic acid showed
that these pieces of stem when grown aseptically on agar medium supplemented
with nutrients and vitamins increased their rubber content if some substances
were added. The results indicated that these isolated stem pieces were able
to synthesize rubber. The substances which caused rubber synthesis were:
leaf extracts of guayule, acetate, acetone, and ß-methyl crotonic acid. These
results substantiate those obtained with guayule seedlings.
Finally ARREGUIN, BoNNER and Woon used radioactive carbon in the form
of C14 labeled acetate, which was either double labeled (prepared biologically by
Clostridium aceticum), or carboxyllabeled acetate. The tagged acetate was fed
in solution to plants growing in pure quartz sand. 72 hours after the addition
of the acetate, the plants were harvested, dried, and the rubber hydrocarbon
extracted. Then the rubber bromide was made and crystallized from various
solvents to constant specific activity. In the case of the double labeled acetate
the rubber formed at its expense contained 3.5 times the specific activity of
the tissue as a whole, indicating that almost all of the carbon atoms of the rubber
had been derived from acetate. The radioactive carbon in the rubber amounted
to a rubber synthesis velocity of 0.3 mg rubber per gm dry weight of tissue in
72 hours.
Handbuch d. Pflanzenphysiologie, Bd. X. 16
242 BARBARiN ARREGUiN: Rubber and latex.

In other experiments with carboxyl labeled acetate of very high specific

activity it was found the following balance of radioactivities shown in table 7.

Table 7. Uptake and distribution of radioactive carbon in guayule.

Fraction
Weight of I S-pe~ill~ : Total % of total
fraction in mg actlvlt~ m I activity in
counts/mm/mg
activity
1 counts/min taken up

Acetate given 19.0 2.5 X 105 4.5 X 106

+
Sterns roots 6.9 75.2" 5.2 X lOS 100.00
Recrist. ruhher bromide. 248.0 20.0 4.9 X 103 0.95
Rubber as hydrocarbon . 70.4 70.3 4.9 X 103 0.95
Benzene sol. non ruhher. 626.0 228.0 1.4 X 106 27.50
Unchanged acetate. BaC03 • 9.0 X 103 1.70
Organic acids. 185.0 44.0 8.2 X 103 1.60

The results in table 7 indicate that only a very small amount of carboxyllabeled
acetate is used in the synthesis of ruhher by guayule plants. Here the specific
activity of ruhher was a little less than that of the entire tissue. The ruhher
bromide purified to constant specific activity by recrystallization from various
solvents, contained only 1% of the total radioactivity. The benzene soluble
fraction contained very appreciable quantities of radioactive carbon, however
in this fraction when the ruhher was excluded still the highest specific activity
was found. The acid fraction also accumulated a sizeable quantity of 0 14 • The
presence of ß-methyl crotonic acid in the organic acid fraction was shown by
the isotope dilution technique which in this case resulted positive.
The mechanism proposed for the biosynthesis of ruhher is given in the
following scheme:
CH3, CH3,
CH3-COOH + )C=O ~ )C=CH-COOH
CH3 CH3

In the experiments described in the work of A:RREGuiN, BoNNER and Woon

a major portion of the acetate went into protein synthesis. The amino aCJids
obtained by acid hydrolysis of the protein fraction of the guayule were fractionated
in a starch column according to MooRE and STEIN and counted in a GEIGER-
MüLLER. The data indicated that most of the 0 14 of the proteinswas present in
leucine, valine, and glutamic and aspartic acids. Thus it can be concluded that
exogenous acetate is used by the guayule in several metabolic pathways, and
only a small portion of it is utilized in ruhher synthesis. ·
All plants synthesize at least one or few isoprenoids like terpenes, carotenoids,
phytol, sterols etc. Some of them are present universally in the higher plants,
and have important functions in the plants. However up to now no real specific
physiological role can be ascribed to rubber, or gutta.
In a recent work by BoNNER, PARKER and MoNTERMoso an account of the
experiments with added precursors to Hevea is discussed. Squares of hark
separated from the rest of the Iatex system by deep cuts after the application
of acetate and ß-methyl crotonate produced 50% more rubber. Furthermore,
growing small Hevea trees in a 0 140 2 atmosphere produced rubber evenly labeled
with 0 14 . It took several hours for the reaction to take place and was suggested
that acetate enters into the metabolic pathway of rubber through its conden-
sation with coenzyme A. The synthesis of ß-methylcrotonate from acetate has
been studied in enzvme extracts of plants by JoHNSTON, RACUSEN and BoNNER.
 Evaluation of the theories on rubber biosynthesis. 243

According to these findings with experimental support which seems quite good,
the sequence of reactions involved in the formation of ß-methylcrotonate are then:
a) CH3-COOH +Co A ~ CH3-CO-Co A
b) 2 CH3-CO-Co A ~ CH3-CO-CH 2-CO-Co A + Co A
c) CH3-CO-CH2-CO-Co A + H 20 ~ CH 3-CO-CH 2-COOH +Co A
CH3" ' ~CH2-COOH
d) CHa-CO-CH 2--CO-OH + CH3-CO-Co A ~ /C
HO H 2-CO-Co A
CH3...._ /CH2-COOH CH3...._
e) /"C( ~ /"C=CH-00-Co A + H 20 + C0 2
HO ~H 2 -CO-Co A CH3

The initial steps in the mechanism of ruhher formation are similar to those in
the hiosynthesis of fatty acids. ß-hydroxy-ß-methyl glutarate isanatural product
present as a necic acid of the alkaloid dierotalme (see ADAMSand VAN DUUREN}
which occurs in Crotalaria dura, and hence has received the name of dicrotalic
acid. This acid is also found as an ester in the flax seed or Linum usitatissimum,
where it was isolated hy KLosTERMAN and SMITH.
The enzymatic reaction leading to ß-hydroxy-ß-methyl glutaric acid has heen
studied in animal tissues hy STADTMAN, DounoROFF and LIPMAN, as well as hy
MILLERD and BaNNER in plant tissues. Reactions d and e have heen studied
experimentally from right to left, using ß-methyl crotonate and carhon dioxide,
and laheling either of these two suhstances with C14 • In either case the product
of the reaction is acetoacetate which greatly exceeds the amount of ß-hydroxy-
ß-methyl glutarate.
In general the path for the formation deviates from that of the fatty acid
metaholism at step c. ß-hydroxy-ß-methyl glutarate was found hy BANDURSKI,
CoYLE and BaNNER (see JoHNSTON, RACUSEN and BaNNER) to he a product
of acetate conversion in plants.
An interesting finding hy JoHNSTON, RACUSEN and BaNNER indicates that
the enzyme systems of flax extracted from the apical sections of the stems,
are ahle to form citrate, acetoacetate, ß-hydroxy-ß-methyl glutarate, and ß-methyl
crotonate from C14 laheled acetate, and that this conversion requires high energy
phosphate in the form of adenosine triphosphate (ATP), and coenzyme A. All
the experimental evidence points to the formation of coenzyme A (Co A) deriva-
tives of ß-hydroxy-ß-methyl glutarate and ß-methyl crotonate. The same enzyme
preparation from flax will produce ß-hydroxy-ß-methyl glutarate from ß-methyl
crotonate and C140 2 as the starting materials, and this, too, has the same require-
ments for ATP and Co A.
The experimental evidence accumulated thus far enahles us to postulate a
hiosynthetic path for the formation of ß-methyl crotonate different from the
condensation of acetone and acetate, which had heen proposed earlier for the
guayule.
Furthermore in the guayule as in lemon grass, enzymes are present capahle
of synthesizing laheled ß-hydroxy-ß-methyl glutarate from C14 laheled acetate.
The participation of the activated form of ß-methyl crotonate with Co A in
ruhher synthesis was made clear from experiments in guayule and Hevea. In
spite of all these findings the mechanism of conversion of ß-hydroxy-ß-methyl
glutarate, or ß-methyl crotonate to ruhher remains ohscure.
The chemical production of tree ruhher has recently heen achieved by FrnE-
STONE and GooDRICH GuLF (see Chem. and Eng. News). Theinfrared and X-ray
Handbuch d. Pflanzenphysiologie, Bd. X. 16a
244 BARBARIN ARREGUIN: Rubber and latex.

analysis of this man-made rubber are identical with the natural rubber. It is
an all-cis 1-4 polyisoprene; the synthesis of it was achieved by polymerization
at low temperatures of isoprenein the presence of suitable catalysts, like Iithium.

Summary.
The facts piled up through experimental work on the biochemistry of rubber
have clarified some of the metabolic reactions involved in the formation of rubber.
It has been established that the precursor of rubber in Hevea and the guayule,
and possibly in all rubber synthesizing plants could arise from acetate. ß-hydroxy-
ß-methyl glutarate and ß-methyl crotonate are intermediates in the biosynthesis
of rubber. It is required now to establish conclusively the identity of these
precursors with the naturally occuring precursors of rubber in plants.
More fundamental work is needed to discover the way plants actually reduce
these c5 intermediates and polymerize them to rubber hydrocarbon.

Addendum.
Plant hormone application to Hevea has been successfully used to induce
higher rubber yields. Comprehensive physiological investigations on the effect
of plant hormones have been made by WIERSUM who has published methods to
prepare these rubber yield-stimulants, and the mode of application, as well as the
results obtained. He has reported an extra yield of 50 kg of rubber in high yielding
Hevea stands following the treatment of 2,4-D and Stimulex.
A thorough testing by applying the paste containing the plant hormones to:
a) bark of Hevea trees, b) partially isolated bark sections with a connecting bark
strip opposite to the tapping cut, c) totally isolated bark areas and d) stumped
trees, achieved by girdling or by removing the top of the trees, led WIERSUM to
conclude that normal trees treated with Stimulex showed Iatex yield increases
over the controls, which Iasted for several months. In girdled trees without hor-
mones a marked decrease in Iatex yield is detected, whereas if the girdled ones are
submitted to Stimulex treatments, they showslight increases during a short period
of time.
In a quantitative way it can be said that, if the control trees are taken as 100%
rubber production, those applied with Stimulex produced 20% more rubber, while
the girdled trees treated showed a slight increase over the corresponding untreated
girdled controls, this latter effect was also found in stumps of Hevea trees.
Restrietion of the rubber drainage areas in hevea by girdling or stumping
results in lower yields. The addition of hormone pastes to bark areas produces
yield increases whereas the application of anti-auxins such as maleic hydrazide
causes as expected much lower yields than those obtained in the controls.
The hormone stimulation in stumped trees causes an increased Iatex flow and
rubber production does not rise, which is contrary to what happens after hormone
stimulation in intact trees where no decrease in dry rubber content (D.R.C.) can
be noticed. Thus in intact trees, hormone application can be broken into two
phenomena: a) an increased rubber production without dilution, and b) increased
rubber production by augmenting the flow of latex (with a lower dry rubber con-
tent) as happens in intensive tapping.
In addition, an accumulation of starch content above the upper rings of com-
pletely isolated bark, and a decrease of starch in the isolated area, was observed
whereas if the bark keeps a connecting strip with the top of the trees, there is
no starch decrease in the trunk.
 Literature. 245

The effects of complete girdling are in agreement with previous findings.

Girdling above and below the tapping results in marked decrease in yields and
leaving a connecting bark strip in the upper girdle gives intermediary results, as
compared with one girdle or with two complete girdles.
A danger of exhaustion of Iatex formation by hormone treatment was not
found even after several months, which emphasizes the economical importance
of this treatments to enhance rubber production in Hevea plantations. The sug-
gestion is made that the hormone produces higher metabolic activity which leads
to a quicker regeneration of the lactiferous tissues responsible for latex pro-
duction. This explains the constancy of the D.R.C. and a net rubber increase
with a more normal growth of the tapped tree, making for more economical
rubber production if this technique is applied with care.
Excellent work on the comparison of particle fractions in the Apocynaceae by
v AN DIE has recently appeared. This author studied 40 species belonging to
40 genera, and he was able to divide the components of latices into several well
defined chemical groups. The latex obtained from the plants was diluted and
cooled to -7° C, a technique which induced floculation. After 4 to 7 days a
coagulate was formed and aserum separated. The cohesion of the coagulates was
greater as the polyisoprene containing compounds increased. In this way theserum
constituents were removed from the Iatex coagulate, and by further dialysis the
water soluble constituents occluded in the coagulate were completely removed.
Proteins, alkaloids, rubber, gutta, triterpenes, tannins, salts of organic acids
and cardiotonic glycosides can all in turn be major constituents of Apocynaceae
latices.
In general the fresh latex coagulate is soluble in benzene to the extent of 98
to 100%.
In some genera of the Apocynaceae family rubber or gutta are present in the
coagulates whereas in others they are absent. In Ficus elastica plants varying
from 3-35 years old, the rubber present in coagulates increased with age from
63.3% up to 95.1 %.
A quantitative distribution of rubber in latex coagulates from various organs
of A pocynaceae plants showed that the polyisoprene content of them is nearly con-
stant provided that the Iatex originated from the same lactiferous system. The
composition of the latex coagulates from the same parts of a singleplant is aproxi-
mately constant.
This comprehensive comparative study of all the polyisoprene containing com-
pounds present in the latex coagulates showed a great variation in the individual
substances. The compounds derived from the isoprene hydrocarbon and pro-
bably related chemogenetically studied in the work of VAN DIE were rubber, gutta
triterpenes, fatty acids, all of which may be derived biosynthetically from acetate
via aceto-acetate.

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