**2. Experimental design and material and methods**

Design of experiment is completely randomized and selection of plants was seasonal based during the work carried out in the laboratory. Plants were raised in the glass house under controlled conditions of light (light (PAR) ranged between 1050 to 1180 μmol m−2 s−1 at 12.00 noon), humidity (80–92%) and temperature (maximum and minimum temperature ranged between 37 and 43°C and 28 and 34°C respectively).

**Sand culture:** Soybean (*Glycine max* var. JS-335), mustard (*Brassica juncea* var. varuna) and linseed (*Linum usitatissimum* var. R-552) were grown in sand culture using the technique developed at the Long Ashton Research Station, Bristol, U.K. [18] and standardized for Indian conditions by Agarwala and Sharma [19]. The composition of nutrient solution used for growing the plants excluding B was: 4 mM KNO3, 4 mM Ca (NO3)2, 2 mM MgSO4, 1.33 mM NaH2PO4, 0.1 mM Fe EDTA, 10 μM MnSO4, 1 μM CuSO4, 1 μM ZnSO4, 0.1 μM Na2MoO4, 0.1 mM NaCl, 0.1 μM CoSO4 and 0.1 μM NiSO4. Boron was supplied as H3BO3 at varying levels as sufficient amount (0.33 mg L−1 B supply) and insufficient (0.033 mg L−1 B supply) amount.

**Seed germination:** Seeds of all crops were first surface-sterilized with 5% (v/v) mercuric chloride solution and washed properly with deionized manesty still water (MSW) before germination. Sterile seeds were then sown in petridishes lined with three fold filter paper in double distilled water at 28°C and 85% relative humidity in seed germinator. The percentage germination of post harvested seeds of all crops was recorded after 48 h.

**Seed protein:** Seed protein was extracted by the method of Sommour [20]. After the harvesting of crops seeds were collected and seed coat was removed and seeds were ground in acetone. The extract was centrifuged 3–4 times at 11,500 xg for 10 min. Portions of air dried seed flour (200 mg) were extracted with water for albunins, 5% NaCl for globulins, 0.1 N NaOH for glutenins and 70% ethanol with 2 drops of mercaptoethanol for prolamines at room temperature. The proteins in the above extracts were estimated by the method of Lowry et al. [21]. The optical density of the reaction mixture was measured on spectrophotiometer at 750 nm. The readings were referred to a standard calibration curve prepared from crystalline bovine serum albumin.

#### **3. Oilseed crops**

Oilseed crops have been the backbone of agricultural economy of India from times immemorial. Today these crops are cultivated on about 26.67 million hectares, with total production of 30.06 million tonnes [22]. This area constitutes approximately one-tenth of the total cultivated area in India. On the oilseed map of the world, India occupies a prominent position, both in regard to acreage and

*Grain and Seed Proteins Functionality*

growth in most plant species. Boron increases flower production and retention, pollen tube elongation and germination and seed and fruit development. The reproductive growth especially flowering, fruit and seed set and seed yield is more effective even at moderate boron insufficiency than vegetative growth [2, 3]. Various reports in many crops exhibited that boron can be inadequate and have a notable impact on yield even when there are no vegetative symptoms of boron insufficiency and supply of boron is also adequate [4, 5]. Shedding of buds, flowers and developing fruits and seeds as well poor fruit/seed quality and poor seed viability are seen in crops grown under inadequate boron at the onset of reproductive blooming [6]. Male sterility and retarded microsporogenesis and pollen fertility in wheat due to poor translocation of boron from vegetative to reproductive parts is the main cause of poor grain yield [7]. Post fertilization development and seed maturation is also influenced by the boron nutrition. Poor germination and vigor of seeds was reported in low boron crops [8]. Increment in phenolic compounds and fall in oil content in seeds of *Sesamum* plants receiving inadequate boron nutrition was spotted by Sinha *et al.* [9]. Chatterjee and Nautiyal [10] reported that boron deprivation in sunflower caused morphological aberrations in seeds and bring down the seed content of non-reducing sugars, starch and oil, even at the commencement of

The demand of boron nutrition at the time of flowering and seed set is much higher in many crops even when boron concentration in vegetative organs are in appropriate amount Increased abnormal seedlings and decreased germination rate during seed germination was observed in boron insufficient seeds [8, 11, 12]. Various workers have reported that there is an enhancement in fruit set and yield with boron foliar fortification [5, 13]. The same was observed during the reproductive stage of sunflower leads to increment in seed yield suggesting involvement of boron in reproductive biology [4] and also qualitative and quantitative improve-

Dordas [15] mentioned the consequences of boron foliar fertilizers on pod development, pod set, seed set, seed yield and yield components such as pod number per inflorescence, seed number per pod, seed development, seed weight, and on seed quality in terms of seed germination and seed vigor and observed that the seed yield was enhanced approximatly 37% and seed germination and seed vigor improved upto 27% in alfalfa as compared to untreated control. They also noticed that the critical boron levels for alfalfa used for forage production is below that for seed production and boron foliar fertilizeration can enhance the seed yield and seed quality of alfalfa grown for seed production. Pandey and Gupta [16] reported improved seed yield and seed vigor in the boron insufficient black gram plants given foliar application of boron. They also reported improved seed quality in terms of storage seed proteins (albumin, globulin, glutenin and prolamin) and carbohydrates (sugars and starch) in black gram received foliar boron fertilizer. The reality of that boron implementation improved seed vigor specifies that seeds with adequate boron nourishment can germinate and produce seedlings with better ability to grow and resist any adverse environmental conditions. This is in agreement with the suggestion that seeds that were developed in plants with adequate nutrient supply show high germination percentage and also have high seed vigor [17].

The essentiality of boron suggests that there is a continuous requirement of it during the entire growth period of plants. There is a critical requirement of boron for reproductive development and seed quality which must be met at the onset of the reproductive phase. Thus boron deficiency is a major factor responsible for low seed yield and quality of oil yielding crops widely cultivated on boron deficient soils world over. The aim of the research work conducted is to put together the effect of boron on reproductive development, production, maturation and nutritive quality

**176**

anthesis.

ment in strawberry fruits [14].

production. Oilseed crops are grown primarily for the oil contained in the seeds. The major world sources of edible seed oils are soybeans, sunflowers, rapeseed, cotton and peanuts. Seed oils from Flax (linseed) and castor beans are used for industrial purposes. Oilseed crops used for experimental work in the present study are:

**Soybean:** The soybean (U.S.) or soya bean (UK) (*Glycine max*) is a species of legume native to East Asia and originated in China. It is also known as a 'miracle crop' with over 40% protein and 20% oil. The plant is classed as an oilseed rather than a pulse. Soybean oil contains significantly greater amount of omega-6 fatty acids in the oil: 100 g of soybean oil contains 7 g of omega-3 fatty acids to 51 g of omega-6: a ratio of 1:7. Flaxseed, in comparison, has an omega-3: omega-6 ratio of 3:1. Soybeans also contain the isoflavones genistein and daidzein, types of phytoestrogen, that are considered by some dietitians and physicians to be useful in the prevention of cancer and by others to be carcinogenic and endocrine disruptive. Soy's content of isoflavones is as much as 3 mg g−1 dry weight. Isoflavones are polyphenol compounds, produced primarily by beans and other legumes, including peanuts and chickpeas [23]. The major unsaturated fatty acids in soybean oil triglycerides are 7% α-linolenic acid (C-18:3); 51% linoleic acid (C-18:2); and 23% oleic acid (C-18:1). It also contains the saturated fatty acids 4% stearic acid and 10% palmitic acid. The soybean seed storage proteins are classified in 8-conglycinin (7S) and glycinin (liS) proteins and multiple genes are responsible for their production [24]. The seed storage proteins of soybean are reported to be devoid of the S-containing essential amino acids, methionine and cysteine. Seed proteins consist of subunits dissimilar in amino acid profile i.e. 8-subunit of 7S protein. Approximately 60% l1S proteins consisting 3–4.5% sulfur amino-acid content and 40% 7S proteins consisting >1% sulfur amino-acid content are present in storage protein of soybean crops [25, 26].

**Mustard:** Rapeseed mustard (*Brassica*) contributes 32% of the total oilseed production in India, and it is the second largest indigenous oilseed crop. This species is of Asiatic origin and grows in southern regions of the Former Soviet Union, the Caucasus, Western and Eastern Siberia, the Far East, Central Asia. Generally it is also distributed in Middle Europe, Asia Minor, Iran, Afghanistan, India, Mongolia, China, Japan. *Brassica juncea* infests all spring crops including grain and tilled crops, and vegetable gardens. This plant is cultivated as an oil crop and for preparation of mustard powder in the south-east of the European part of Russia, in Ukraine, Belorussia and the North Caucasus. Seeds of *B. juncea* contain 25–30% fatty non-drying oil and glycoside sinigrine. Oilcake is used for preparation of mustard powder. Leaves are used for food in salads, they contain up to 150 mg of ascorbic acid. *B. juncea* is a good bee plant. Transgenic Indian mustard are also useful in phytoremediation of heavy metals and metalloids such as Cd, Cr, Cu, Mn, Zn, Pb, Hg, As, and Se [27, 28]. Indian mustard (*Brassica juncea*), are suitable target species for this strategy because of having a large biomass production, a relatively high trace element accumulation capacity [29], and can be genetically engineered [28]. Mustard oil is a healthy cooking medium because of low saturated fatty acids (8%), high monosaturated fatty acids (70%) and α-linolenic acid (10%) [30]. The mustard seeds are rich in lysine with considerable amounts of sulfur containing methionine and cysteine amino acids. Thus the mustard seed protein is the excellent source of human nutrition [31]. The most common seed storage proteins found in the cotyledons is cruciferin and napin. Cruciferin proteins are of 12S legumin-like globulin protein with mol wt 300-360 kDa and napins belongs to 2S prolamin-super family albumin with mol wt 12.7–20.3 kDa. These both proteins are differing in molecular forms, amino acid profiles as well as physico-chemical and biological properties.

**179**

**4. Boron**

tion in different forms- BO2

*Impact of Inadequate Concentration of Boron in Seed Storage Proteins Content in Oilseed Crops*

**Linseed:** Flax also known as common flax or linseed (*Linum usitatissimum*) is a member of the family Linaceae. It is native to the region extending from the eastern Mediterranean to India and was probably first domesticated in the Fertile Crescent. Flax seeds contain high levels of lignans and omega-3 fatty acids. Lignans may benefit the heart; possess anti-cancer properties and studies performed on mice found reduced growth in specific types of tumors. Initial studies suggest that flaxseed taken in the diet may benefit individuals with certain types of breast and prostate cancers [32, 33]. Linseed oil is a rich source of linolenic acid (40–60%), an omega 3 fatty acid which has anti-inflammatory action in the treatment of arthritis. It also helps in lowering down the cholesterol level in mammals. Lignan present in oil has anti-carcinogenic effect [34]. Legumin- like proteins of mol wt 320 ± 20 and subunit mol wt of 55, 54.5,50,45,43 and 41 is the characteristic feature of majority of linseed seed proteins. These proteins contain non –covalently bound carbohydrates with pI- values 4.5 to 8. The 2- mercaptoethanol reduces the legumin-like protein polypeptides into acidic and basic subunits with mol wt 25–40 and mol wt 18–22 respectively. Albumin –like proteins contained major subunit with mol wt 25 and minor subunit with mol wt 11 [35]. Sammour et al. [35] also reported that metabolic or antimetabolic activitiy is not performed by both types of subunits and their storage functioning is found to be due to abundance in nitrogenous amino acids.

Boron is a member of the subgroup III of metalloids and has intermediate properties between metals and non-metals. The boron atom is small and has only three valencies and has a strong affinity for oxygen. Boron is present in soil solu-

, BO3, H2BO3

molecule, its permeability coefficient for transport across the lipid bilayer is several orders of magnitude higher than that of ions. The physical and chemical properties of boron and its complexes are unique and highly varied. Under physiological conditions and in the absence of interaction with bio-molecules, boron exists as boric acid (B[OH]3) or borate anion (B[OH4]-). Boric acid is a very weak acid, with a pKa of 9.24 a cytoplasmic pH (pH 7.5), more than 98% of boron exists in the form

apoplast (pH 5.5), greater than 99.95% of boron is in the form of B(OH)3 and less

react with many kinds of biological molecules and under normal biological conditions available boron binding molecules exceed the concentration of free boron. An understanding of boron binding reactions is therefore central to an understanding

Boron is widely distributed in lithosphere and hydrosphere, boron concentration ranging from 5 to 10 mg kg−1 in rocks [38], 3–30 μg kg−1 in rivers [39] and ~ 4.5 mg L−1 in ocean [40]. Boron is found mostly in the topsoil. Dry weather reduces moisture in the topsoil and boron uptake by the plant, causing boron deficiency. Even high rainfall areas witness leaching out of borosilicate from the soil, which leads to boron deficiency. Boron deficiency is the most widespread micronutrient deficiency in agricultural crops in world including India. The symptoms of boron deficiency are observed when boron content in soil comes down to 5 to 25 mg ha−1. Aluminum hydroxide adsorbs large amounts of soluble boron, making the soil acidic and causing boron deficiency. Soils containing a high proportion of

Some specific symptoms found in plants grown under inadequate boron nutrition are: 'Top sickness' of tobacco; 'Corky core' or 'internal cork' or 'drought spot' of

−

−

−

and [B (OH) 4]. As an uncharged

[36]. At pH values found in the

. Boric acid and borate however can readily

− , B4O7 −

of free B(OH)3 and less than 2% exists as B(OH)4

than 0.05% is in the form of B(OH)4

organic matter are less deficient in boron.

of boron physiology [37].

*DOI: http://dx.doi.org/10.5772/intechopen.95873*

*Impact of Inadequate Concentration of Boron in Seed Storage Proteins Content in Oilseed Crops DOI: http://dx.doi.org/10.5772/intechopen.95873*

**Linseed:** Flax also known as common flax or linseed (*Linum usitatissimum*) is a member of the family Linaceae. It is native to the region extending from the eastern Mediterranean to India and was probably first domesticated in the Fertile Crescent. Flax seeds contain high levels of lignans and omega-3 fatty acids. Lignans may benefit the heart; possess anti-cancer properties and studies performed on mice found reduced growth in specific types of tumors. Initial studies suggest that flaxseed taken in the diet may benefit individuals with certain types of breast and prostate cancers [32, 33]. Linseed oil is a rich source of linolenic acid (40–60%), an omega 3 fatty acid which has anti-inflammatory action in the treatment of arthritis. It also helps in lowering down the cholesterol level in mammals. Lignan present in oil has anti-carcinogenic effect [34]. Legumin- like proteins of mol wt 320 ± 20 and subunit mol wt of 55, 54.5,50,45,43 and 41 is the characteristic feature of majority of linseed seed proteins. These proteins contain non –covalently bound carbohydrates with pI- values 4.5 to 8. The 2- mercaptoethanol reduces the legumin-like protein polypeptides into acidic and basic subunits with mol wt 25–40 and mol wt 18–22 respectively. Albumin –like proteins contained major subunit with mol wt 25 and minor subunit with mol wt 11 [35]. Sammour et al. [35] also reported that metabolic or antimetabolic activitiy is not performed by both types of subunits and their storage functioning is found to be due to abundance in nitrogenous amino acids.

#### **4. Boron**

*Grain and Seed Proteins Functionality*

protein of soybean crops [25, 26].

study are:

production. Oilseed crops are grown primarily for the oil contained in the seeds. The major world sources of edible seed oils are soybeans, sunflowers, rapeseed, cotton and peanuts. Seed oils from Flax (linseed) and castor beans are used for industrial purposes. Oilseed crops used for experimental work in the present

**Soybean:** The soybean (U.S.) or soya bean (UK) (*Glycine max*) is a species of legume native to East Asia and originated in China. It is also known as a 'miracle crop' with over 40% protein and 20% oil. The plant is classed as an oilseed rather than a pulse. Soybean oil contains significantly greater amount of omega-6 fatty acids in the oil: 100 g of soybean oil contains 7 g of omega-3 fatty acids to 51 g of omega-6: a ratio of 1:7. Flaxseed, in comparison, has an omega-3: omega-6 ratio of 3:1. Soybeans also contain the isoflavones genistein and daidzein, types of phytoestrogen, that are considered by some dietitians and physicians to be useful in the prevention of cancer and by others to be carcinogenic and endocrine disruptive. Soy's content of isoflavones is as much as 3 mg g−1 dry weight. Isoflavones are polyphenol compounds, produced primarily by beans and other legumes, including peanuts and chickpeas [23]. The major unsaturated fatty acids in soybean oil triglycerides are 7% α-linolenic acid (C-18:3); 51% linoleic acid (C-18:2); and 23% oleic acid (C-18:1). It also contains the saturated fatty acids 4% stearic acid and 10% palmitic acid. The soybean seed storage proteins are classified in 8-conglycinin (7S) and glycinin (liS) proteins and multiple genes are responsible for their production [24]. The seed storage proteins of soybean are reported to be devoid of the S-containing essential amino acids, methionine and cysteine. Seed proteins consist of subunits dissimilar in amino acid profile i.e. 8-subunit of 7S protein. Approximately 60% l1S proteins consisting 3–4.5% sulfur amino-acid content and 40% 7S proteins consisting >1% sulfur amino-acid content are present in storage

**Mustard:** Rapeseed mustard (*Brassica*) contributes 32% of the total oilseed production in India, and it is the second largest indigenous oilseed crop. This species is of Asiatic origin and grows in southern regions of the Former Soviet Union, the Caucasus, Western and Eastern Siberia, the Far East, Central Asia. Generally it is also distributed in Middle Europe, Asia Minor, Iran, Afghanistan, India, Mongolia, China, Japan. *Brassica juncea* infests all spring crops including grain and tilled crops, and vegetable gardens. This plant is cultivated as an oil crop and for preparation of mustard powder in the south-east of the European part of Russia, in Ukraine, Belorussia and the North Caucasus. Seeds of *B. juncea* contain 25–30% fatty non-drying oil and glycoside sinigrine. Oilcake is used for preparation of mustard powder. Leaves are used for food in salads, they contain up to 150 mg of ascorbic acid. *B. juncea* is a good bee plant. Transgenic Indian mustard are also useful in phytoremediation of heavy metals and metalloids such as Cd, Cr, Cu, Mn, Zn, Pb, Hg, As, and Se [27, 28]. Indian mustard (*Brassica juncea*), are suitable target species for this strategy because of having a large biomass production, a relatively high trace element accumulation capacity [29], and can be genetically engineered [28]. Mustard oil is a healthy cooking medium because of low saturated fatty acids (8%), high monosaturated fatty acids (70%) and α-linolenic acid (10%) [30]. The mustard seeds are rich in lysine with considerable amounts of sulfur containing methionine and cysteine amino acids. Thus the mustard seed protein is the excellent source of human nutrition [31]. The most common seed storage proteins found in the cotyledons is cruciferin and napin. Cruciferin proteins are of 12S legumin-like globulin protein with mol wt 300-360 kDa and napins belongs to 2S prolamin-super family albumin with mol wt 12.7–20.3 kDa. These both proteins are differing in molecular forms, amino acid profiles as well as physico-chemical and biological

**178**

properties.

Boron is a member of the subgroup III of metalloids and has intermediate properties between metals and non-metals. The boron atom is small and has only three valencies and has a strong affinity for oxygen. Boron is present in soil solution in different forms- BO2 − , B4O7 − , BO3, H2BO3 − and [B (OH) 4]. As an uncharged molecule, its permeability coefficient for transport across the lipid bilayer is several orders of magnitude higher than that of ions. The physical and chemical properties of boron and its complexes are unique and highly varied. Under physiological conditions and in the absence of interaction with bio-molecules, boron exists as boric acid (B[OH]3) or borate anion (B[OH4]-). Boric acid is a very weak acid, with a pKa of 9.24 a cytoplasmic pH (pH 7.5), more than 98% of boron exists in the form of free B(OH)3 and less than 2% exists as B(OH)4 − [36]. At pH values found in the apoplast (pH 5.5), greater than 99.95% of boron is in the form of B(OH)3 and less than 0.05% is in the form of B(OH)4 − . Boric acid and borate however can readily react with many kinds of biological molecules and under normal biological conditions available boron binding molecules exceed the concentration of free boron. An understanding of boron binding reactions is therefore central to an understanding of boron physiology [37].

Boron is widely distributed in lithosphere and hydrosphere, boron concentration ranging from 5 to 10 mg kg−1 in rocks [38], 3–30 μg kg−1 in rivers [39] and ~ 4.5 mg L−1 in ocean [40]. Boron is found mostly in the topsoil. Dry weather reduces moisture in the topsoil and boron uptake by the plant, causing boron deficiency. Even high rainfall areas witness leaching out of borosilicate from the soil, which leads to boron deficiency. Boron deficiency is the most widespread micronutrient deficiency in agricultural crops in world including India. The symptoms of boron deficiency are observed when boron content in soil comes down to 5 to 25 mg ha−1. Aluminum hydroxide adsorbs large amounts of soluble boron, making the soil acidic and causing boron deficiency. Soils containing a high proportion of organic matter are less deficient in boron.

Some specific symptoms found in plants grown under inadequate boron nutrition are: 'Top sickness' of tobacco; 'Corky core' or 'internal cork' or 'drought spot' of

#### **Figure 1.**

*Various symptoms in plants received inadequate boron nutrition (data source online except 11 and 12 these are original data from our experiment).*

apple; 'Water core' of turnip'; 'Hard fruit' of Citrus; 'Yellows' of alfa alfa; 'Hen and chicken' of grapes; 'Heart rot' of sugarbeet; 'Stem crack' of celery; 'Hollow stem' of cauliflower and broccoli and 'Tipburn' of chinese cabbage ([41, 42]; **Figure 1**). Boron deficiency also creates an unfavorable situation for the pollen-stigma interaction and limits fertilization leading to poor reproductive yield ([42]; **Figure 1**). Boron takes part in various vital functioning of crops like translocation of sugars, metabolism of RNA, protein, indole acetic acid, phenols, ascorbate, osmotic and oxidative stress etc. [1, 43, 44].
