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III. Methods of Determining Boron in Plants and Soils
BORON NUTRITION OF CROPS
However, difficulties are experienced in automating this method because both
the reagent and the borate-chromotropate complex are sensitive to light (James
and King, 1967). Chromogenic reagents, such as quinalizarin and carminic acid,
are specific and sensitive for determining B. But their use in automated procedures appears to be limited because they must be used in a concentrated H2S04
medium (Willis, 1970; Lionnel, 1970). The curcumin method has been modified
and was found to be rapid and simple (Fiala, 1973). However, reagents such as
curcumin suffer from the disadvantage that the values for B determined by such
methods vary with the amount and salt content of the material analyzed (Williams
and Vlamis, 1970). Other spectrophotometric methods are based on the conversion of sample B into fluoroborate (BF;) in an H,S04-HF system, which is then
extracted as a colored complex with Azure-C (or methylene blue) into dichloroethane and determined spectrophotometrically (Weir, 1970).
Procedures other than colorimetric include the determination of B by means of
spectrographic and atomic absorption spectrophotometric methods. However,
these methods are not as sensitive to B as the color reagents, and therefore their
use has been limited.
A new color-developing reagent, azomethine-H, originally used by Russian
workers for determining B in organic compounds, was first used by Basson el al.
(1969) for determining B in plant materials. Since that time the method has
achieved prominence for determiring B in soil, compost, and manure (Wolf,
1971); it will be dealt with in detail in this section.
A . BORON IN PLANTS
Dry and wet ashing are the most common methods for extracting B from
plants. The carmine method of Hatcher and Wilcox (1950), also used with some
modifications by Gupta (1967a), and the curcumin method of Williams and
Vlamis (1970) have been the most common methods for determining B in plant
extracts during the past 10 to 20 years. However, with the advent of the
azomethine-H method in the late sixties this color reagent has become very
popular. Wolf (1971) extended its application in the determination of B from
compost and manures using a colorimetric technique. Recently, J. A. Smith and
D. A. Tel, University of Guelph, Ontario, Canada, have automated the
azomethine-H colorimetric method, using the technique developed by Basson et
al. (1969). These scientists made some modifications by adding 4 g of NaOH per
liter of EDTA reagent to give a pH 4.9 to the waste coming from the AutoAnalyzer system. The only interferences with this method are due to the presence of Al, Cu, and Fe in the plant extracts. Such interference is easily overcome
by use of a 0.25 M EDTA (disodium salt) solution (Basson et al., 1969). Sippola
and Ervio (1977) reported that the recoveries of added B ranged from 94 to 108%
in plant extracts, when determined by the azomethine-H method using spec-
Coefficient of Variabitity in the Boron Content of Some Plant leaf Tissues from Various Loeations
in Prince Edward Island, Canada
(Medicago sativa L.)
(Trifolium prarense L.)
(Phleum pratense L.)
Wheat (boot stage tissue)
(Triricum aestivwn L.)
(Daucus carora L.)
(Beta vulgaris L.)
(Brassica napobrassica, Mill)
(Brassicu oleraceu var.
(Zea mays L.)
(Nicoriana rabacum L.)
" Values (ppm) are averages of four replicate determinations.
BORON NUTRITION OF CROPS
trophotometry; these values were similar to those obtained by the carmine
method. The azomethine-H method has also been tested by the author; results
based on quadruplicate analyses of a number of plant materials indicated good
reproducibility based on low coefficient of variability (Table 11). This method is
very simple and does not require concentrated acids. It is convenient for use on
an AutoAnalyzer and is recommended for precise determinations of large numbers of samples on a routine basis.
B. AVAILABLE BORON IN SOIL
Until 10 years ago, the only method for extracting available B in soils involved
refluxing the soil with hot water for a period of 5 minutes (Berger and Truog,
1939). However, this method was time-consuming and involved several steps,
including the addition of K2C03to the filtrate, evaporation, and ignition of the
residue. The residue, after being dissolved in H2S04, was triturated. Subsequently, Gupta (1967b) developed a rapid and satisfactory method whereby the
soils could be extracted by boiling with water directly on a hot plate. The filtrates
thus obtained could be analyzed directly, using the then commonly known
color-developing reagent, carmine.
Since the development of this simplified method, researchers in various parts
of the world (Wolf, 1971; John, 1973) have used direct extraction of soil with hot
water. The determination of hws B in soil by using azomethine-H as outlined by
Wolf (1971) is probably the most suitable technique. The common interferences
due to Cu and Fe in the extract can be eliminated as outlined under the previously
described method for plants. Wolf (1974), however, reported that for soils high
in A1 the use of sufficient tetrasodium salt of EDTA (rather than disodium salt)
was necessary for complete elimination of interference due to Al. With this
method, precautions should be taken to prevent interference due to organic
products in the water and soil extracts by maintaining uniform quantities of
charcoal in the soil and in the water. The use of much larger amounts of charcoal
than are recommended for soil or water can result in a considerable loss of B
from the sample (Wolf, 1974). With the precautions recommended by Wolf, the
azomethine-H method should be quite satisfactory in determining B in hot-water
extracts of soil.
Azorobacrer chroococcurn was considered as a possible microbiological indicator for B availability in soils (Gerretsen and de Hoop, 1954). This method is
based on the fact that Azotobucter requires B, with up to 8 ppm being required in
soils for its normal development. However, owing to the complicated behavior of
soils, the microbiological determination of B by the Azofobacfer method has
been considered unsatisfactory (Bradford, 1966).
UMESH C. GUFTA
C. TOTAL BORON IN SOIL
There are very few studies on the methods for determining total B in soils. A
procedure for extracting total B from a soil fused with Na2C0, was used earlier
by Berger and Truog (1939) and subsequently described by Wear (1965). This
method is laborious and time-consuming, and it requires an adjustment of the pH
of the extract obtained from the fused mass. Large volumes of methyl or ethyl
alcohol are required to precipitate sodium sulfate formed in the extract. Furthermore, several evaporations are needed before B can be determined from such
extracts. A modified procedure was developed by Gupta (1966) whereby the
fused soil mass was extracted with 6 N HCl. The amount of B in the resulting
extract could be determined directly by using carmine as the color-developing
reagent. No interferences were encountered, the percentage of recovery of B
added to the soil was good, and the results were reproducible. For large numbers
of determinations the method could be automated by using azomethine-H with
some minor modifications.
IV. Role of Boron in Plants
There is perhaps less precise information available on the role of B in plants
than for any other essential micronutrient. The functions of B in plants remained
almost obscure prior to the mid-1950s. The biochemical role of B is as yet not
well understood, and, unlike other generally recognized micronutrients, it has
not been shown to be part of an enzyme system (Jackson and Chapman, 1975).
The objective of this section is to discuss briefly recent information obtained on
the role that B plays in the growth and development of plants.
Some of the chief topics to be discussed here include the effect of B on
translocation of sugars, on root extension and meristematic tissues, on the
pyrimidine biosynthetic pathway, and on ATPase activity.
Van de Venter and Currier (1977) found that callose accumulates in the tissues
of B-deficient bean (Phaseolus vulgaris L.) and cotton (Gossypium hirsuturn L.)
plants. Sieve plates in the phloem of B-deficient beans were characterized by
heavy plugs of callose, whereas the sieve plates of cotton were essentially unaffected. Since translocation of I4C was drastically reduced in both plants, it was
suggested that deposition of callose in B-deficient plants is a secondary effect of
cellular damage. Birnbaum ef al. (1977) found that cotton ovules callus when B
is lacking in the medium.
The research of Sisler et al. (1956) indicated that B enhances uptake and
translocation of sugars and is implicated in carbohydrate metabolism. They proposed a micronutrient union with sugars, giving an ionizable sugar-borate complex that moves more readily through cellular membranes than does sugar alone.
BORON NUTRITION OF CROPS
Deficient tomato (Lycopersicon esculentum, Mill .) plants were found to translocate more sugar when 50 ppm of B was added with sucrose through a cut petiole
than when sucrose was applied alone. Subsequent studies by Dugger and Humphreys (1960) implied a direct involvement of B in the enzymatic reactions of
sucrose and starch synthesis. It has also been suggested that B deficiency possibly causes reduced synthesis of uridine diphosphate glucose (Birnbaum et al.,
Weiser et al. (1964) reported that B does not enhance sugar translocation in
plants, but it does enhance the foliar uptake of sucrose applied to the leaves.
They concluded that this phenomenon of enhanced foliar uptake of sucrose has
given rise in the past to the erroneous conclusion that B enhances sugar translocation.
Zapata (1973) found that sugar cane (Saccharurnoficinarum L.) plants receiving only traces of B suffered growth and quality losses without developing visual
B-deficiency symptoms. Lack of B lowered sucrose production in leaves and
significantly altered the rates of sugar transport in sugar cane storage tissues
(Zapata, 1973). In sugar beets the sucrose content of the storage roots started to
decrease at about the same point at which limiting B resulted in a drop in yield
(Vlamis and Ulrich, 1971).
The earliest morphological symptoms of B deficiency in mung bean
(Phaseolus uureus L.) appear to be a slowdown in root extension, followed by a
degeneration of meristematic tissue, possibly due to a repressive effect of B
deficiency on cell division (Jackson and Chapman, 1975). Results of Robertson
and Loughman (1974) indicated that it is unlikely that responses associated with
B deficiency are caused by interference with cell division, but they may be
related to the role of B in the metabolism, transport, or action of auxin-type
hormones in broad beans (Vicia fubu L.). Whittington (1959) found that
B-deficient field bean roots had enlarged apices and fewer cells than the normal
B-sufficient roots. Investigations of Kouchi and Kumazawa (1976) on tomato
root tips indicated that a lack of B distorted the shape and arrangement of cortical
cells and resulted in an abnormal accumulation of a “lipid-like substance.”
Also, there was an abnormal development of Golgi apparatus, which seemed to
be related to the irregular thickening of cell walls.
Cohen and Lepper (1977) established that cessation of root elongation of intact
squash (Cucurbitupepo L.) plants is an early result of B deficiency. They noted
that the ratio of cell length to cell width ranged from a low of 0.8 in B-sufficient
root meristems to a high of 3.0 in root meristems grown in a B-deficient nutrient
solution for 98 hours. It was concluded that a continuous supply of B is not
essential for cell elongation but is required for maintenance of meristematic
Bioassays showed that extracts of substances similar to indoleacetic acid
(IAA) taken from B-deficient roots were more inhibitory to the growth of bean-