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ZINC SEED PRIMING IMPROVES STAND ESTABLISHMENT, TISSUE ZINC CONCENTRATION AND EARLY SEEDLING GROWTH OF CHICKPEA.

Byline: A. Ullah, M. Farooq, M. Hussain, R. Ahmad and A. Wakeel

Keywords: Deficiency, toxicity, germination rate, root length, seedling dry weight.

INTRODUCTION

Zinc (Zn) is an essential micronutrient for plant growth; its deficiency decreases the yield of many crops. It is major micronutrient disorder which limits the chickpea (Cicerarietinum L.) productivity (Ahlawatet al. 2007), as it is required for pollen functionality and fertilization (Pandey et al. 2006). In chickpea, the deficiency of Zn reduces the rate of photosynthesis (Khan et al. 2004), delaysthe crop maturity and reduces the yield and the water use efficiency at whole plant level (Khan et al. 2003). Zinc deficiency in Pakistani soils has been reported since decades and about 70% of Pakistan's agricultural soils are Zn deficient (Hamid and Ahmad 2001).Chickpea is mostly grown in southern Punjab and deficiency of Zn is more severe in southern Punjab due to sandy nature of soil (Ullah et al. 2019) with extent of Zn deficiency ofabove 75% (Maqsoodet al. 2015).Deficiency of Zn affects the chickpea more severely compared with cereals (Khan 1998).

Chickpea varieties also differ for response to the deficiency of Zn (Khan1998; Ahlawat et al. 2007). Chickpea growth and development is badly affected owing to Zn toxicity as Zn above the needed level behaves as heavy metals (like lead and cadmium) and hinders the development and growth of plants (Ali et al. 2000). For instance, high dose of Zn (300 mg) decreased the root, shoot growth and dry weight of wheat (Triticum aestivum L.) (Glinska et al. 2016).In another study, the application of Zn (above 0.1 ZnCl2 and 0.5 M ZnSO4) was found toxic and affected the germination and seedling growth of wheat (Rehman et al. 2015). Zinc application at 10 mM decreased the germination up to 75% and cause reduction in gibberellic acid and zeatin contents in chickpea (Atici et al. 2005). Zinc can be applied to crop plantsviafoliar and soil application and with seed treatments (priming and coating) (Farooq et al. 2012; Haider et al. 2018).

In soil application, the uniform delivery of micronutrients is difficultand most of the nutrients may get fixed (Ryanet al.2013). To counter the micronutrients deficiency, foliar application is an option, but it is inefficient if not applied timely. It is expensive, requirerepetitive spraysand expertise as well (Takkaret al. 1986). However, Znseed treatmentis an economical and easy option (Farooqet al. 2018).Seed priming is a controlled pre-sowing hydration technique in which seeds are soaked in water or in any solution and then dried back to their original weight (Farooq et al. 2006).Seed priming with nutrients at higher concentrations inhibits the germination of crops e.g., barley (Hordeumvulgare L.) (Zelonka et al. 2005) and wheat (Rehmanet al. 2015; Rehman and Farooq 2016).Therefore, for wide-scale application of nutrients through seed priming, optimizingthe levels of nutrients is needed.

To the best of our knowledge, no information is available regarding the optimization of Zn seed priming in desi and kabuli chickpea. Therefore, this study was conducted to optimize differentconcentrations of Zn seed priming to improve stand establishment, tissue Zn concentration and seedling growth of bothdesi and kabulichickpea.

MATERIALS AND METHODS

Plant material: Seeds ofkabuli chickpea cv. Noor-2013 were collected from Pulses Research Institute, AARI, Faisalabad, Pakistan whereas seeds of desi chickpea cv. NIAB-CH-2016 were obtained from the Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan.

Experimental design and treatment details: This study was consisted of four independent experiments conductedinthe Allelopathy Laboratory, Department of Agronomy, University of Agriculture, Faisalabad, Pakistan during 2016. The experimentaltreatments were executed in completely randomized design in factorial arrange and repeated four times.The experiments were carried out in petri plates (90 mm x 1.5 mm) and in sand filled pots (150 mm x 90 mm). The growth conditions were maintained at 25 +- 2AdegC throughout the experimentation.The first two experiments were pre-optimization experiments and the Zn was applied as seed priming at 0.01, 0.05, 0.1, 0.5 and 1.0 M Zn for both petri plates and sand filled pots using zinc sulphate as source. In experiment, conducted in petri plates, both chickpea types' seeds were uniformly placed between the layers of moist filter papers. In the second experiment, seeds of both chickpea types were sown in plastic pots filled with river sand (500 g pot-1).

There was no germination in both chickpea types at[greater than or equal to] 0.01 M Zn in both medium (Figure 1) (data not given). Based on the result of pre-optimization experiments, Zn concentrations were reduced to (0.01, 0.001 and 0.0001 M Zn) for third and fourth experiments(Figures 2 and 3). For both chickpea types, seeds at 12% moisture content weresoaked in aerated solution using commercial ZnSO4.7H2O(with 33% Zn) as source, for eight hours. An aquarium pump was used to provide aeration. In all experiments, dry seed were taken as control. In sand filled pots and petri plates, eight seeds were sown. In petri plates, seeds exhibited 2 mm radicles (length) were counted as germinated. While in pots, coleoptile appearance above the sand was scored as seedling emergence. After the constant germination the seedlings were thinned to five.

After fifteen days of sowing the seedlings were harvested for growth traits(shoot length, root length, numbers of secondary roots per plant and seedling dry weight) and seedling Zn concentration determination.

Stand establishment, plant growth and seedling Zn concentration: The experiments were visited daily to record germination/emergence count till constant scores. Mean germination/emergence time (MGT/MET) was calculated using the formula of Ellis and Roberts (1981). Fifteen days after sowing, seedlings were harvested to record seedling shoot and root length, number of secondary roots and seedling dry weight. The seedling shoot and root length was measured with measuring tap while the numbers of secondary roots were counted from the same plants to record the numbers of secondary roots per plant. Seedling dry weight was determined by drying in oven at 70AdegC till constant weight. Zinc was estimated by grinding and digesting the roots and shoots samples in di acid (HClO4:HNO3 1:2 v/v) on a digestion plate and Zn was determined using the atomic absorption spectrophotometer(Perkin Elmer, CA, USA)(Prasad et al. 2006).

Statistical analysis: The experimental data were subjected to analysis using statistical package Statistix 8.1 (Analytical Software, USA). The difference among treatments means was compared using Least Significant Difference (LSD) test at 5% probability level (Steel et al. 1997). Microsoft-Excel Program was used to calculate the Pearson correlation and graphical presentation of data.

Table 1. Effect of different concentrations of zinc seed priming on the mean germination time, shoot and root length of chickpea types (Petri plate experiment)

Zn application###Chickpea type###Chickpea type###Chickpea type

###Kabuli Desi###Mean(T)###Kabuli###Desi###Mean(T)###Kabuli###Desi###Mean(T)

###Mean germination time###Shoot length (cm)###Root length (cm)

###(days)

Control###2.51###2.26###2.39A###3.14###2.61###2.88AB###1.58bcd###2.68b###2.13

SP (0.01 M Zn)###1.83###1.89###1.86B###0.48###1.38###0.93C###0.69d###1.14cd###0.92

SP (0.001 M Zn)###1.40###1.55###1.47B###3.23###4.08###3.66A###1.62bcd###4.68a###3.15

SP (0.0001 M Zn)###1.56###1.66###1.61B###2.16###2.28###2.22B###1.48bcd###2.14bc###1.81

Mean (C)###1.82###1.84###2.25###2.59###1.34###2.66

LSD (p a$? 0.01)###0.41###0.81###1.33

Table 2. Effect of different concentrations of zinc seed priming on the numbers of secondary roots and seedling dry weight of chickpea types (Petri plate experiment)

###Chickpea type###Chickpea type

Zn application###Kabuli###Desi###Mean(T)###Kabuli###Desi###Mean(T)

###Number of secondary roots###Seedling dry weight (mg)

Control###06###05###05AB###381###475###428B

SP (0.01 M Zn)###01###03###02B###213###276###244C

SP (0.001 M Zn)###08###10###09A###475###599###537A

SP (0.0001 M Zn)###04###08###06A###327###525###426B

Mean (C)###05###06###349B###468A

LSD (p a$? 0.01)###3.81###C=57; T=81

Table 3. Effect of different concentrations of zinc seed priming on the mean emergence time, shoot and root length of chickpea types (Sand filled pot experiment)

###Chickpea type###Chickpea type###Chickpea type

Zn application###Kabuli###Desi###Mean(T)###Kabuli###Desi###Mean(T)###Kabuli###Desi###Mean(T)

###Mean emergence time (days)###Shoot length (cm)###Root length (cm)

Control###3.52###3.41###3.46 B###27.6ab###21.9bcd###24.7###11.6###15.4###13.5 AB

SP (0.01 M Zn)###4.13###4.33###4.23 A###02.4e###16.9ab###09.7###01.7###11.6###06.7 B

SP (0.001 M Zn)###2.63###2.98###2.80 B###29.3a###25.5abc###27.4###16.9###18.3###17.6 A

SP (0.0001 M Zn)###3.05###3.31###3.18 B###19.4cd###23.9abc###21.6###12.8###13.6###13.2 AB

Mean (C)###3.33###3.51###19.7###22.1###10.7###14.7

LSD (p a$? 0.01)###0.67###6.3###6.8

Table 4. Effect of different concentrations of zinc seed priming on the numbers of secondary roots and seedling dry weight of chickpea types (Sand filled pot experiment)

###Chickpea type###Chickpea type

Zn application###Kabuli###Desi###Mean (T)###Kabuli###Desi###Mean (T)

###Number of secondary roots###Seedling dry weight (mg)

Control###15ab###12b###14###425ab###400b###412

SP (0.01 M Zn)###02c###12b###07###325b###375b###350

SP (0.001 M Zn)###20a###17ab###18###650a###525ab###587

SP (0.0001 M Zn)###13b###14ab###13###457ab###500ab###478

Mean (C)###12###14###433###481

LSD (p a$? 0.01)###7.0###240

Table 5. Correlation of some stand establishment and growth traits with seedling dry weight of chickpea types in petri plate and sand filled pot experiments (n=4)

Variables###Petri plate experiment###Sand filled pot experiment

###Kabuli chickpea###Desi chickpea###Kabuli chickpea###Desi chickpea

Mean emergence time (days)###-0.16###-0.43###-0.94**###-0.83**

Shoot length (cm)###0.94**###0.88**###0.77*###0.91**

Root length (cm)###0.89**###0.85**###0.90**###0.66*

Numbers of secondary roots###0.99**###0.94**###0.88**###0.91**

RESULTS

Petri plates

Stand establishment, plant growth and Seedling Zinc Concentration: Seed priming with Zn significantly affected the mean germination time (MGT), shoot length (SL), number of secondary roots (NSRs),and seedling dry weight (SDW) of both (kabuli and desi) chickpea (Table 1 and 2). Moreover, the interaction of Zn seed priming x chickpea types was significant only for root length (RL) (Table 1). However, chickpea typesonly differedsignificantly for SDW (Table 2).

Seed primingwith 0.001 M Zn solution reduced the MGT of both chickpea types but was statistically similar with 0.01 and 0.0001 M Zn solution. Maximum SL was recorded with 0.001 M Zn solution while minimum with 0.01 M Zn solution in both chickpea types (Table 1). Maximum RL was recorded with 0.001 M Zn solution in desi chickpea, while minimum was with 0.01 M Zn solution in kabuli chickpea (Table 1). The maximum NSRs were recorded with 0.001 and 0.0001 M Zn and minimum with 0.01 M Zn solution in both chickpea types (Table 2). The maximum SDW was recorded with 0.001 M Zn, while minimum with 0.01 M Zn solution in both chickpea types. Among the chickpea types; maximum SDW was produced with desi chickpea compared to kabuli (Table 2).

In case of seedling Zn concentration, maximum seedling Zn concentration was measured with 0.01 M Zn which was toxic for both chickpea types (Figure 4aand 1). While, Zn seed priming concentration at which chickpea types performed best was 0.001 M Zn solution for both chickpea types (Figure 2a, 3aand 4a). However, minimum seedling Zn concentration was recorded where no Zn was applied in both chickpea types (Figure 4a).

Sand filled pots

Stand establishment, plant growth and Seedling Zinc Concentration: Seed priming with Znsignificantly affected the mean emergence time (MET), shoot length (SL), root length (RL), numbers of secondary roots (NSRS) and seedling dry weight (SDW) of both (kabuli anddesi) chickpea types (Table 3 and 4). Moreover, the interaction of Zn seed priming x chickpea types was significant for SL, NSRs, and SDW whereas non-signifucant for MET and RL. However, chickpea types didn't significantly differed for the Zn seed priming (Table 3 and 4). Seed priming with 0.001 and 0.0001 M Zn solution reduced the MET in both chickpea types which is statistically similar to control. The maximum SL was recorded with 0.001 M Zn while, minimum with 0.01 M Zn solution in kabulichickpea (Table 3).

Maximum RL was recorded with 0.001 M Zn solution in both chickpea types (Table 3). Maximum NSRs were recorded with 0.001 M Zn while, minimum with 0.01 M Zn solution in kabuli chickpea. Moreover, higher SDW was recorded with 0.001 M Zn solution in kabuli chickpea while, minimum with 0.01M Zn in both chickpea (Table 4). Correlation analysis showed that shoot and root length, and numbers of secondary roots per plant had strong positive correlation with seedling dry weight of both chickpea types grown in petri plates or sand filled pots under different concentrations of Zn seed priming. However, seedling emergence had strong negative correlation with seedling dry weight of both chickpea types grown in sand filled pots under different concentrations of Zn seed priming (Table 5).

Maximum seedling Zn concentration was measured with 0.01 M Zn solution in both chickpea which was statistically similar with 0.001 M Zn solution in desi chickpea (Figure 4b). However, minimum seedling Zn concentration was recorded where no Zn was applied in kabuli chickpea (Figure 4b).

DISCUSSION

In this study, the optimization and potential of Zn seed priming was evaluated for the early stand establishment, tissue Zn concentration and improving seedling growth of both (desi and kabuli) chickpea. Seed priming with Zn improved the emergence/germination and seedling development of chickpea (Tables 1-4) as priming of seedschange the physiology of embryo and activates the hydrolytic enzymes by which emergence occur at a rapid pace than normal seedling emergence (Bam et al. 2006).Moreover, germination/emergence and seedling growth was improved by the application of Zn in priming solution(Tables 1-4)as Zn is involved in the radicle development and in the early stages of coleoptile growth and in auxin synthesis (Ozturket al. 2006).

Seedling dry weight was increased with Zn seed priming due to early and uniform emergence/germination, which in result improved the seedling growth (Tables 1-4) as the deficiency of Zn decreases the root growth in chickpea (Khan 1998).Moreover, the improvements in seedling growth of both chickpea types with Zn seed priming might be due to significant improvement in shoot and root length and numbers of secondary roots per plants as seedling dry weight had strong positive correlation with growth traits (Table 5). Zinc seed priming at 0.01 M Zn was proved toxic whichsuppressed the germination/emergenceand seedling growth of chickpea (Tables 1-4).Root growth is restricted at higher concentration of Zn as Zn toxicity suppresses the process of cell division (Prasad et al. 1999) (Tables 1-4). Moreover, the root and leaf development were reduced at higher concentration of Zn owing to substantial decrease in NADPH production in chloroplasts (Mousavi 2011).

The different chickpea varieties behave different to the deficiency of Zn (Ahlawatet al. 2007). The chickpea types differedfor response to Zn levels and the Zn above the required level wastoxic for both chickpea types (Figures 1-4). The higher application of Zn inhibit germination and reduced the growth and development of chickpea (Tables 1-4; Figure 1) as application of Zn at higher concentration(1.0 and 10 mM Zn)causedelay and impeded the germination in chickpea due to decrease in gibberellic acid and zeatin in germinating seeds (Aticiet al. 2005).The over doze of Zn inhibits the growth and development of plants (Ali et al. 2000) owing to its involvement in metabolic processes linked with normal development (Gadallah and El-Enany 1999).

Heavy metals including Zn decrease the germination of seeds (Ali et al. 2000) as heavy metals in high concentration disturb the physiological and biochemical processes, for example, injure cell membranes, affect the enzyme activity, damage photosynthetic apparatus and rate, and break the protein synthesis (Monniet al. 2001; Aticiet al. 2003). Among the tested chickpea types, desichickpea performed better in stand establishment and growth compared with kabuli chickpea (Tables 1-4). However, regarding germination/emergence kabulichickpea was better than desi chickpea (Tables 1-4). Application of micronutrients above the required level behaves as heavy metal and cause stress in plants. Chickpea is very sensitive to Zn deficiency and toxicitythus, there is need to optimize the Zn concentrationsfor seed priming for field application to improve the growth, productivity and nutritional value of grain.

In conclusion, Zn seed priming with 0.001 M Zn solution performed best in improving the early seedling germination/emergence, tissue Zn concentration and growth of both chickpea types compared with other Zn seed priming concentrations. However, at high concentration [greater than or equal to] 0.01 M Zn solution seedlings germination and growth suppressed which indicatedZn toxicity for chickpea.

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