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III. Methods of Determining Boron in Plants and Soils

III. Methods of Determining Boron in Plants and Soils

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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.


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

Location 2

Location 1

Plant species


(Medicago sativa L.)

Red clover

(Trifolium prarense L.)


(Phleum pratense L.)

Wheat (boot stage tissue)

(Triricum aestivwn L.)


(Daucus carora L.)


(Beta vulgaris L.)


(Brassica napobrassica, Mill)

BNS& sprouts

(Brassicu oleraceu var.

gemmifera Zenker)


(Zea mays L.)


(Nicoriana rabacum L.)

B (ppm)"

Coefficient of


B (ppm)

Coefficient of





















I .6


















1 .o


I .O

" Values (ppm) are averages of four replicate determinations.



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.


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).




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.


28 1

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-

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