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IMPROVING SEED YIELD OF BLACK GRAM(VIGNA MUNGO L. VAR. DPU-88-31)THROUGH FOLIAR FERTILIZATION OFZINC DURING THE REPRODUCTIVE PHASENalini Pandey a & Bhavana Gupta aa Plant Nutrition and Stress Physiology Laboratory, Department ofBotany , University of Lucknow , Lucknow , IndiaPublished online: 30 Jul 2012.

To cite this article: Nalini Pandey & Bhavana Gupta (2012) IMPROVING SEED YIELD OFBLACK GRAM (VIGNA MUNGO L. VAR. DPU-88-31) THROUGH FOLIAR FERTILIZATION OF ZINCDURING THE REPRODUCTIVE PHASE, Journal of Plant Nutrition, 35:11, 1683-1692, DOI:10.1080/01904167.2012.698349

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Journal of Plant Nutrition, 35:1683–1692, 2012Copyright C© Taylor & Francis Group, LLCISSN: 0190-4167 print / 1532-4087 onlineDOI: 10.1080/01904167.2012.698349

IMPROVING SEED YIELD OF BLACK GRAM (VIGNA MUNGO L. VAR.

DPU-88-31) THROUGH FOLIAR FERTILIZATION OF ZINC DURING

THE REPRODUCTIVE PHASE

Nalini Pandey and Bhavana Gupta

Plant Nutrition and Stress Physiology Laboratory, Department of Botany, University ofLucknow, Lucknow, India

� The response of zinc (Zn) deficiency on reproductive yield and recovery through foliar applicationof Zn was determined in black gram, an important edible legume in India. Results revealed thatfoliar spray of Zn [0.01 and 0.5% zinc sulfate (ZnSO4)] improved not only the flowering, pollenproducing capacity, pollen viability, stigma-receptivity and pollen-stigma interaction but also theyield parameters like number, size and weight of pods and seeds. Zinc deficiency induced changes inpollen grain structure, activity of esterase and poor stigmatic exudation leading to improper pollen-stigma interaction, which seem to be the main cause of poor fertility and decreased seed yield. Foliarapplication of 0.5% ZnSO4 after bud formation was most beneficial not only for reproductive yieldbut also seed Zn content.

Keywords: black gram, pollen grains, pollen-stigma interaction, reproductive yield,stigma, Zn

INTRODUCTION

More than 50% of Indian agricultural land is zinc (Zn) deficient. Thewide spread problem of Zn deficiency might be related to the introductionof high yielding varieties, application of bulky manures, imbalanced use offertilizers and low Zn uptake. Several environmental factors like soil pH,temperature, light intensity, organic matter and soil moisture also plays animportant role in limiting plant-available Zn (Singh et al., 2005). Analysis ofnearly 250,000 soil samples and 2,500 plant samples collected from differentstates in India showed that 48% of the soil samples and 44% of the plant sam-ples contained inadequate levels of Zn (Singh, 2007). Zinc has been shown

Address correspondence to Nalini Pandey, Plant Nutrition and Stress Physiology Laboratory, Depart-ment of Botany, University of Lucknow, Lucknow-226007, India. E-mail: nalini pandey@rediffmail.com

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to play critical roles in protection against toxic effects of reactive oxygenspecies and plant reproductive development (Cakmak, 2000; Pathak et al.,2005; Pandey et al., 2006, 2009). A large number of proteins bind/require Znfor their function and about 40% of the Zn binding proteins are transcrip-tion factors which are essential for gene expression during flowering suchas floral primordial initiation (Nakagawa et al., 2004), anther and pollensize, pollen viability, stigmatic morphology (Payne et al., 2004), exudationand receptivity, seed set and viability of seed (Papi et al., 2002). Studies haverevealed that stigma from Zn deficient plants showed lack of stigmatic exu-dation, required for pollen tube hydration and pollen tube growth throughthe style, leading to improper pollen-stigma interaction and finally reducedseed set (Pandey et al., 2006, 2009). The seeds produced contained low Zncontent, which showed poor germination and seedling growth.

Seeds with low Zn content further aggravate the Zn malnutrition in hu-man beings. Thus it is necessary to develop improved fertilizers technology(biofortification) and higher yielding varieties as a prerequisite not only forimproving yield but also producing Zn enriched seed. Agronomic biofortifi-cation strategy especially by foliar spray has gained impetus to improve seedyield as well as Zn concentration in seeds. The positive effect of Zn sprayson nutritional status, seed set, and yield were supported by other research(Thalooth et al., 2006; Mousavi et al., 2007). Cakmak (2009) reported re-cently that foliar application of Zn is an efficient way to maximize seed Znconcentration. Black gram (Vigna mungo) commonly known as ‘urd’, is awidely consumed edible legume grown throughout India, both as a pureand as inter-mixed crops. Among the pulses, black gram ranks fourth inproduction and acreage. It covers an area of about 3,011,300 hectares withthe annual production of 1,295,400 tons throughout India. Black gram seedcontain 26% protein (three-fold higher than cereals), 55% carbohydrates,32% starch and about 10% dietary fibers (both insoluble and soluble) andform an important source of vegetarian diet. In present study therefore aneffort was made to identify the concentration of Zn most suitable for foliarapplication of Zn to improve the quantitative and qualitative yield of blackgram under Zn deficient conditions.

MATERIALS AND METHODS

Plant Culture and Experimental Design

Seeds of black gram (Vigna mungo cv. DPU-88-31) plants were raised inpurified sand and supplied with nutrient solution containing 4 potassium ni-trate (KNO3); 4 calcium nitrate [Ca(NO3)2], 2 magnesium sulfate (MgSO4),1.33 sodium phosphate (NaH2PO4), 0.33 boric acid (H3BO3), 0.1 iron (Fe)ethylenediaminetetraacetic acid (EDTA) (in mM), 10 manganese sulfate(MnSO4), 1 copper sulfate (CuSO4), 0.1 sodium molybdate (Na2MoO4),

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0.1 sodium chloride (NaCl), 0.1 cobalt sulfate (CoSO4), and 0.1 nickel sul-fate (NiSO4) (in µM) and Zn supplied as zinc sulfate (ZnSO4) at control(1.0 µM) and deficient (0.1 µM) levels of Zn supply (Sharma, 1996).

At the time of germination, plants were separated in two sets. The 1st setwas supplied with normal (1.0 µM) and 2nd set was supplied with deficientZn (0.1 µM). During the initiation of flowering, the 2nd set of Zn deficientplants were further divided into seven sets. While one lot of Zn-deficientplants continued to receive deficient supply, the remaining six sets of Zndeficient plants were sprayed with two concentration of Zn as 0.01 and 0.5%ZnSO4 at three different stages of reproductive development i.e. before(prior to flowering), and after bud formation and prior to anthesis. Eachfoliar spray was done three times and the total treatments were as under:

1st set: Zn sufficient plants Control2nd set: Zn deficient plants without any foliar spray ZnD3rd set: ZnD plants given foliar spray of 0.01% ZnSO4 before bud

formationZn1+F1

4th set: ZnD plants given foliar spray of 0.01% ZnSO4 after budformation

Zn1+F2

5th set: ZnD plants given foliar spray of 0.01% ZnSO4 prior toanthesis

Zn1+F3

6th set: ZnD plants given foliar spray of 0.5% ZnSO4 before budformation

Zn2+F1

7h set: ZnD plants given foliar spray of 0.5% ZnSO4 after budformation

Zn2+F2

8th set: ZnD plants given foliar spray of 0.5% ZnSO4 prior toanthesis

Zn2+F3

Light (PAR) ranged between 740 to 980 µmol m−2 s−1 at 12.00 noon, relativehumidity (RH) ranged between 68–98% at 9.30 A.M. and minimum andmaximum temperature ranged between 24–30 and 30–40◦C, respectively,during the period of experiment.

Tissue and Seed Zn Concentration

Concentration of Zn in leaves, stem, root and seed was determined inwet acid digests [nitric acid (HNO3): perchloric acid (HClO4)] by atomicabsorption spectrophotometer (Perkin Elmer Analyst 300; Perkin Elmer,Waltham, MA, USA).

Pollen Producing Capacity

Mature anthers per flower were removed from five unopened flowers(bud) of five plants and a homogenous suspension was prepared by gently

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crushing each anther in 10% (v/v) glycerol. The number of pollen grains inthe suspension was counted under compound microscope.

Pollen Viability

The pollen viability was determined by germinating the pollen grains ina culture medium containing 10% sucrose, 0.01% boric acid, 0.03% calciumnitrate, 0.02% magnesium sulfate and 0.01% potassium nitrate (Brewbakerand Kwack, 1963). Scoring was done of 10 sets of 20 pollen grains each, fromeach treatment.

Enzymes

Stigma from 10–20 flowers was gently excised and placed in the reactionsolution for 10–20 min at 25–35◦C in a humid chamber. Staining of acidphosphatase, esterase and peroxidase was done as described by Pandey et al.(2006).

Pod and Seed Yield

Ten plants per treatment in triplicates were tagged before the foliarapplication and the number of flowers formed were counted and recorded.When the pods became dry, the number, length and weight of pods andseeds formed were also measured.

Seed Viability

Seed viability was performed at room temperature (25◦C) in petri disheslined with moist filter paper. Two replicates of 50 seeds for each treatmentwere used. Germinated seeds with emerged radicles (at least 2 mm) werecounted after 4d.

Statistical Analysis

The data have been evaluated by means of ANOVA. The mean valuesand the standard error (±) have been presented along with significant dif-ferences (P ≤ 0.05) in tables and figures.

RESULTS

After 15 days of receiving deficient Zn supply, Zn deficient plants showeddepression in vegetative growth, condensation of internodes, suppression ofbranching and reduction in leaf size. The leaves of Zn deficient plants alsodeveloped some visible symptoms, such as marginal chlorosis of leaflets,which later turned necrotic. With foliar application of Zn supplied as ZnSO4

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a reversal of effects of Zn deficiency was observed. Plants at Zn1+F3 andZn2+F2 showed an appreciable recovery in visible appearance of Zn defi-ciency. Compared to control, Zn deficient plants showed delay in floweringby 10 days. The number of flowers and pods also decreased in plants receiv-ing deficient Zn supply. As Zn deficiency continued, the number of flowerbuds was reduced drastically and the terminal buds of the inflorescencefailed to open. However the foliar application of Zn at all the concentrationat different stages promoted the floral initiation and reproductive develop-ment, being more marked at Zn1+F3 and Zn2+F2 levels. Foliar spray of Znto the deficient plants increased the vegetative dry matter yield of the plantsto varying extent, but the increase was not more than that of control plants.The effect of foliar spray on dry matter yield was most pronounced in plantsat Zn2+F2 treatments (Table 1).

Zinc deficient plants had lower concentration of Zn, which further in-creased to varying extent with foliar application of Zn. The increase in Znconcentration due to foliar spray was particularly marked in leaves and stembut it was not increased significantly in root. Foliar spray at Zn2+F2 levelsshowed a marked increase in Zn concentration in leaves which was morethan that of the control plants (Table 1).

TABLE 1 Effect of foliar Zn supply on dry matter yield and tissue Zn of black gram plants (Vigna mungoL. var. DPU-88-31)

Dry matter yield: g plant−1

Zn supply Leaves Stem Top Root Whole plant

Control a0.97 ± 0.019 a1.59 ± 0.011 a2.56 ± 0.011 a0.195 ± 0.001 a2.75 ± 0.081ZnD b0.66 ± 0.012 b0.43 ± 0.009 b1.09 ± 0.001 b0.063 ± 0.011 b1.153 ± 0.061Zn1+F1 c0.57 ± 0.021 c0.63 ± 0.013 c1.20 ± 0.013 b,c0.060 ± 0.011 b1.26 ± 0.095Zn1+F2 b0.69 ± 0.024 d0.84 ± 0.010 d1.53 ± 0.015 b,d0.080 ± 0.011 c1.61 ± 0.061Zn1+F3 d0.78 ± 0.013 e0.96 ± 0.014 e1.74 ± 0.021 e0.095 ± 0.011 d1.835 ± 0.039Zn2+F1 b,d0.73 ± 0.021 f0.73 ± 0.033 d1.46 ± 0.013 b0.064 ± 0.011 c1.524 ± 0.051Zn2+F2 e0.97 ± 0.014 e1.05 ± 0.032 f2.02 ± 0.019 e0.092 ± 0.011 e2.112 ± 0.063Zn2+F3 d0.78 ± 0.015 d0.80 ± 0.054 g1.58 ± 0.021 b,d0.084 ± 0.011 c1.664 ± 0.090

Tissue Zn: µg g−1 dry weight

Zn supply Leaves Stem Root Seed

Control a52.6 ± 1.51 a29.00 ± 2.94 a26.56 ± 0.026 a33.00 ± 1.09ZnD b12.78 ± 1.24 b11.67 ± 2.99 b10.40 ± 0.031 b21.58 ± 1.11Zn1+F1 c37.80 ± 1.60 c17.02 ± 1.67 c6.20 ± 0.012 a34.02 ± 0.95Zn1+F2 d40.30 ± 1.44 c17.40 ± 3.87 c6.20 ± 0.017 c36.30 ± 0.89Zn1+F3 e46.40 ± 1.31 d24.00 ± 1.28 c6.40 ± 0.056 d38.20 ± 0.67Zn2+F1 e45.18 ± 1.56 e26.60 ± 3.75 c6.10 ± 0.061 c35.90 ± 1.14Zn2+F2 a53.04 ± 1.12 f28.65 ± 1.03 d8.20 ± 0.039 e42.80 ± 1.17Zn2+F3 f48.30 ± 1.81 e26.60 ± 2.96 c,d7.40 ± 0.056 d38.40 ± 1.21

Foliar Zn supplied as ZnSO4: Zn1 = 0.01%; Zn2 = 0.5%. Foliar spray: F1 = before bud formation,F2 = after bud formation, F3 = prior to anthesis. Differences between group means with different lettersin the same column are significant (P ≤ 0.05), control (1.0 µM Zn).

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TABLE 2 Effect of foliar Zn supply on size of anthers, pollen producing capacity (PPC), pollen sizeand pollen and seed viability in black gram (Vigna mungo L. var. DPU-88-31)

Zn supplyAnther sizel×b (µm)

PPC (grainanther−1)

Pollen size(µm)

Pollen viability(% germination)

Seed viability(% germination)

Control a876 ± 1.56 a587 ± 2.18 a92.2 ± 2.02 a86 ± 1.89 a89 ± 1.78ZnD b489 ± 1.53 b289 ± 1.16 b82.1 ± 2.09 b52 ± 1.34 b46 ± 1.26Zn1+F1 c692 ± 1.06 c492 ± 3.02 c80.3 ± 2.25 c61 ± 1.52 c73 ± 1.53Zn1+F2 d772 ± 1.35 d456 ± 2.99 d85.6 ± 2.98 d78 ± 2.18 d83 ± 1.89Zn1+F3 e645 ± 1.09 e424 ± 2.18 b,e83.6 ± 3.89 a83 ± 2.08 e97 ± 2.41Zn2+F1 c696 ± 1.36 f443 ± 2.27 b,e83.8 ± 3.82 e71 ± 1.93 c75 ± 1.23Zn2+F2 f798 ± 1.54 g485 ± 1.89 f87.5 ± 3.19 d79 ± 2.18 a92 ± 1.65Zn2+F3 g625 ± 1.21 e421 ± 1.34 e84.5 ± 2.83 e74 ± 1.87 f79 ± 2.13

Foliar Zn supplied as ZnSO4: Zn1 = 0.01%; Zn2 = 0.5%. Foliar spray: F1 = before bud formation, F2= after bud formation, F3 = prior to anthesis. Differences between group means with different lettersin the same column are significant (P ≤ 0.05), control (1.0 µM Zn).

Supply of additional Zn as a foliar spray to Zn deficient plants increasedthe Zn concentration in seeds. Seeds of plants at Zn1+F3 and Zn2+F2 levelshowed a marked increase in seed Zn concentration that was more than theseeds of control plants. At Zn2+F2 the seeds contained 42.80 µg Zn g−1 ascompared to 33.00 µg Zn g−1 in seeds of control plants. (Table 1).

The size of the anthers and pollen producing capacity (PPC) of theZn deficient plants was markedly reduced as compared to the controlplants (Table 2). Supply of additional Zn, as a foliar spray showed in-crease in anther size and pollen producing capacity (PPC) being moremarked in plants sprayed with 0.5% ZnSO4 during anthesis (Zn2+F2)(Table 2). Zinc deficient pollen grains showed smaller size, which was par-tially increased after foliar application of Zn (Table 2). The pollen grains ofthe Zn deficient plant were not only smaller in size than the normal pollengrains but also showed decrease in their viability, as observed by acetocarminestaining and in vitro germination. Staining of pollen grains for viability re-vealed that while pollen grains of control plants were stained deeply, that ofZn deficient plants failed to stain (Figure 1 A–B). The percent germinationof pollen grains in vitro was more in pollen grains obtained from normalZn than from deficient Zn plants (Figures 1C and 1D). Pollen grains fromZn deficient plants either took more time in pollen germination or devel-oped short pollen tube as compared to normal pollen grains. Many pollengrains of Zn deficient plants also exhibited bursting of the pollen tubes (Fig-ure 1D). The viability of pollen grains was reversed by foliar supply of Zn(Table 2).

In Zn deficient plants the stigmatic surface lacked exudation due towhich less pollen grain was seen adhering to the stigma (Figures 1E and 1F).Foliar application of Zn at all stages along with all concentrations increasedthe exudation at stigmatic region. As a consequence of Zn deficiency,the activity of acid phosphatase and peroxidase was increased and that of

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FIGURE 1 Acetocarmine staining revealed deeply stained viable pollen grains of A) control and B)poorly stained (arrow) non-viable pollen grains of Zn deficient black gram plants. Pollen grains ofC) control and D) Zn deficient plants are showing in vitro germination. Profusely germinating pollengrains (arrow) on stigma of E) control plants and F) pollen grains of Zn deficient plants showing poorgermination (arrow). Histochemical localization of G, H) acid phosphatase, I, J) esterase and K, L)peroxidase in control (G, I, K) and Zn deficient (H, J, L) plants.

esterase was decreased in the stigmatic surface of Zn deficient plants (Figures1G and 1L). Compared to control plants (Figures 1G and 1K), acid phos-phatase and peroxidase stained more deeply (Figures 1H and 1L) whereasesterase stained lighter in the stigma of Zn deficient plants (Figure 1J).Deeper staining of esterase indicated higher activity in stigma of controlplants (Figure 1I).

Zinc deficiency showed more severe effect on reproductive yield thanvegetative yield. The number, length and weight of pods were reduceddue to Zn deficiency. Zinc deficient plants developed merely half numberof pods per plant, which were smaller in length, grey in color, hairy andshrunken in appearance and reduced in weight as compared to the pods ofcontrol plants. Foliar application of ZnSO4 increased pod yield appreciably,at all levels of supply, the most pronounced increase was in plants suppliedZn1+F3 and Zn2+F2. A significant increase in pod length was also observedin foliar sprayed plants. Increase in pod number and pod length togethermay contribute to an increase in pod weight (Figure 2). As in case of podyield, the seed yield also showed appreciable decrease in Zn deficient plants.Application of foliar Zn increased the seed yield appreciably. The increasein seed number, size and weight was appreciable at Zn1+F3 and Zn2+F2levels (Figure 2).

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FIGURE 2 Effect of foliar Zn supply on A) pod number, B) pod length, C) pod weight, D) seed number,E) seed size and F) seed weight in black gram (Vigna mungo L. var. DPU-88-31). Foliar Zn suppliedas ZnSO4: Zn1 = 0.01% and Zn2 = 0.5%. (Foliar spray: F1 = before bud formation, F2 = after budformation, F3 = prior to anthesis). Bars indicate SE ± mean (n = 3). Asterisks (∗) denote significance atLSD P ≤ 0.5%.

DISCUSSION

Black gram plants subjected to Zn deficiency showed a more drastic ef-fect in reproductive yield than the biomass yield. Higher requirement ofZn for reproductive development than vegetative yield has been reportedby several workers (Sharma et al., 1987; Pandey et al., 2006, 2009). Presentstudy revealed that Zn deficiency reduced the number of flowers per plant,which might be related to poor growth, and development of plants. Theflowers of Zn deficient plants showed poor PPC (pollen producing capacity)of anthers and poor viability of pollen grains as observed by in vitro germi-nation. Takatsuji et al., (1992) reported that TFIIIA-type Zn-finger proteinshave been shown to control the development of floral parts by controllingcell division in petunia. Under Zn deficiency, the growth and differentia-tion of sporogenous tissues and ovules remained arrested. The role of Znin reproductive development has gained importance as anther-specific Znfinger (Kobayashi et al., 1998; Kapoor et al., 2002) and Zn finger polycombproteins (Grossniklaus et al., 1998) are suggested to be involved in microand mega-gametogenesis respectively, resulting in impaired embryogenesisand destroyed protoplasmic integrity. Thus loss of pollen viability and pistilreceptivity under Zn deficient condition might be a reason for low seed yield.

Acid phosphatase and peroxidase are hydrolytic enzymes and their ac-tivity increased under Zn stress. Peroxidase may play a direct role in the

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regulation of pollen tube growth and pollen-stigma interaction by metabo-lizing the phenolic compound in pistil (Bredemeijer, 1984). Esterases areinvolved in pectin hydrolysis, pollen tube entry into the pectin-cellulose cellwall of stigma and facilitate the growth of pollen tube within the transmittingtissue of style for successful fertilization (Hiscock et al., 2002). Decrease inesterase activity under Zn deficiency could also inhibit pollen germinationon stigma. The decrease in the activity of esterase and lack of stigmaticexudates, which are required for pollen adhesion, and germination in Zndeficient plants has been reported earlier (Pandey et al., 2006, 2009).

Supply of additional Zn as a foliar spray before and after bud formationand during anthesis at two different concentrations (0.01 and 0.5%) provedvery beneficial for reproductive yield in black gram. ZnD plants given foliarspray of 0.01 and 0.5% before bud formation increased the number of flow-ers per plant by stimulating the male and female gametogenesis. Providingadequate concentration of Zn through foliar application appears to stimulatesporogenous tissue production, leading to increased pollen grain numberper anther. Similarly in stigma, additional supply of Zn increased stigmaticexudation, esterase activity and was beneficial in facilitating pollen-stigma in-teraction, which ultimately resulted in proper germination of pollen grainsand normal development of pods and seeds in Zn deficient foliar sprayedplants. Thus foliar applied Zn increased the pod and seed yield of plants,the maximum increase being observed in ZnD plants given foliar spray of0.5% ZnSO4 after bud formation stage (Zn2+F2) followed by foliar spray of0.01% ZnSO4 during anthesis (Zn1+F3).

In the present study we observed that foliar application not only in-creased seed yield but also increased seed Zn concentration that contributedgreatly to better seed viability and seedling vigor especially under stressfulcondition. Furthermore, high seed Zn concentration would also protectthe germinating seed and seedling from disease infestation and increasingtolerance to different environmental stress factors such as drought, hightemperature, salinity and micronutrient (Zn) deficiency.

The present study provides increasing evidence to show that foliar ap-plication of Zn under deficient conditions is a highly effective and a verypractical way to maximize uptake and accumulation of Zn in seed legumesfor enhanced seed production and high seed Zn density. In conclusion,foliar spray favors a new tool of agricultural practices, through which Znconcentration required for maximum yield production can be supplied tothe plants.

ACKNOWLEDGMENTS

This study was financially supported by a grant from Uttar Pradesh Coun-cil of Science and Technology, Lucknow, Project No. CST/Biotech/RS7/D-862.

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