Basmati Rice Quality Enhancement by Zinc Fertilization and Green Manuring on a Sub-tropical Inceptisol in Indo-Gangetic Plains of India

Basmati (aromatic) rice is premier rice grown in north-western India and Pakistan. This rice is preferred for their long and slender kernels which expand 3-4 times in length and remain fluffy and are well known all over the world, especially in the Middle East and South Asia for their long fluffy grains on cooking. Paddy soils are usually deficient in organic matter because of high temperature and moisture, which causes rapid decomposition of organic matter. The importance of leguminous green manure crops in improving soil fertility, and soil physical properties received increasing attention. Also, the zinc (Zn) deficiency in soils is prevalent worldwide, especially in high pH calcareous soils. No reports were available on combining green manuring crops and Zn fertilization on productivity, Zn content and kernel quality of Basmati rice. Therefore, the current investigation was undertaken to quantify the combined effects of summer green manuring crops and zinc fertilization on productivity, Zn content and kernel quality of Basmati rice in summer green manuring-Basmati rice cropping system. A field study was therefore conducted for two years (2009 and 2010) on a sandy clay-loam soil (typic Ustochrept) at the research farm of the ICAR-Indian Agricultural Research Institute, New Delhi, India. The experiments were conducted in split plot design, keeping three green manuring crops viz. Sesbania aculeata (Dhaincha), Crotalaria juncea (Sunhemp), and Vigna unguiculata (Cowpea) and one summer fallow treatment as main-plot treatments and six Zn sources viz. control (no Zn application), ZnSO4·7H2O (21% Zn), ZnSO4·H2O (33% Zn), ZnO (82% Zn), ZnSO4·7H2O + ZnO (50% + 50%) and EDTA-chelated Zn (12% Zn) in sub-plots and was replicated thrice. The experiments in both the years were conducted with a fixed lay-out plan on the same site. The results showed that incorporation of green manures along with zinc (Zn) fertilization increased grain and straw yield, enhanced Zn concentrations and improved the kernel quality before and after cooking in Basmati rice ‘Pusa Basmati 1’. The application of EDTA-chelated Zn (12% Zn) was the best in terms of grain and straw yield and Zn concentrations in grain and straw and kernel quality before and after cooking Basmati rice. Application of ZnSO4·7H2O (21% Zn) was the second-best treatment followed by ZnSO4·H2O (33% Zn) and ZnSO4·7H2O + ZnO (50% + 50%). Application of ZnO (82% Zn) had least effect in increasing the studied parameters. The lowest values were observed with control (no Zn application). Among the summer green manuring crops, incorporation of Sesbania aculeata (Dhaincha) was found to be the best over Crotalaria juncea (Sunhemp), Vigna unguiculata (Cowpea) and summer fallow in terms of grain and straw yield, Zn concentrations in grain and straw and kernel quality before and after cooking in Basmati rice. Zn fertilization with EDTA-chelated Zn (12% Zn) lead to 25.91 and 21.26% higher grain yield; 60.66 and 82.14% Zn-denser grains; with 13.33 and 10.92% increase in head rice recovery in Basmati rice over control (no Zn application) during 2009 and 2010, respectively.


Introduction
Basmati (aromatic) rice is the premier rice grown in north-western India and Pakistan. This rice is preferred for their long and slender kernels which expand 3-4 times in length and remain fluffy. The varieties of Basmati rice differ greatly in kind and intensity of aroma. The aroma in rice is due to chemical diacetyl 1-pyrolline (V. P. ). These type of rice are well known all over the world, especially in the Middle East and South Asia for their long fluffy grains on cooking, a desirable characteristic for the dish Biryani or Pulao made by cooking rice with vegetables, mutton or chicken and flavoured with special oriental spices (Shivay et al., 2010). Traditional Basmati rice in India's Doon valley is tall indicas yielding 1.5 to 2.0 tonnes per hectare and lodge on heavy fertilization. Therefore, attempts were made at the ICAR-Indian Agricultural Research Institute, New Delhi to develop Basmati rice varieties having high yielding qualities of semi-dwarf indicas rice and having better quality aromas than the traditional one. Considering the great demand of Basmati rice globally, the researchers made concerted research efforts at the ICAR-Indian Agricultural Research Institute, New Delhi, India to develop rice varieties having both Basmati traits and high yield potential. This involved convergent breeding and simultaneous selection at field and laboratory for the complex inherited key characteristics of Basmati quality (extra-long slender grain, excessive elongation on cooking, aroma and ideal physico-chemical properties of starch) and high yield potential of new semi-dwarf rice varieties (Shivay et al., 2010). Pusa Basmati-1 resulted from this research, which took about 25 years and now occupies the largest area in north-western India and fetches about the US $ 500 million in the international market (Siddiq, 2006). 'Pusa Basmati-1' is high yielding Basmati rice with long and slender kernels as per classification by Jennings et al. (1979). Basmati rice varieties developed from IARI now covered about 60% of the total area under rice in north-western India, which is estimated at 5 million hectares.
Paddy soils are usually deficient in organic matter because of high temperature and moisture, which causes rapid decomposition of organic matter (Mohammed et al., 2005). The application of green manures to the soil is considered a good management practice in any agricultural production system because it can increase cropping system sustainability by reducing soil erosion and ameliorating soil physical properties, by increasing soil organic matter and fertility levels (Mandal et al., 2003), by increasing nutrient retention and by reducing global warming potential (Robertson et al., 2000). Leguminous and non-leguminous plants are used in the production of green manures. Leguminous plants form symbiotic associations with Rhizobium bacteria to fix N 2 . This fact causes the green manures, which their principal component are leguminous plant debris, supply to the important soil amounts of N in relation to the green manures obtained from non-leguminous plants.
The importance of leguminous green manure crops in improving soil fertility, and soil physical properties received increasing attention (Whitbread et al., 2000;Ray & Gupta, 2001). The improvement in physical soil conditions as a result of build-up of organic matter by incorporation of green manure or crop residue is associated with a decrease in bulk density, increase in total pore space, water-stable aggregates and hydraulic conductivity of the soil (Tejada et al., 2008a(Tejada et al., , 2008b. Fast-growing leguminous green manures with their adaptability to fit in rice-based cropping patterns and their ability to fix atmospheric nitrogen may offer opportunities to increase and sustain productivity and income in the rice-based cropping systems (Yadvinder-Singh et al., 1991). Green manures enhance organic matter which is the most important benefit credited to green manures. The positive effect of green manures on paddy yield has been reported by Sharma and Prasad (1999). Hemalatha et al. (1999) observed that in-situ incorporation of cowpea before transplanting of rice increased the grain yield by 18% and straw yield by 16% and the quality of rice.
Similarly, Bhatti et al. (1983) reported that Sesbania green manuring substantially improved grain yield to 72%. Incorporation of green manures before transplanting rice can ameliorate the Fe and Zn deficiency by promoting reduced condition and improving other physico-chemical properties of soil. Sesbania aculeata (Dhaincha), Crotalaria juncea (Sunhemp) and Vigna unguiculata (Cowpea) are some of the important leguminous green manuring crops for north-western Indian region. It is expected that regular incorporation of green manuring crops before transplanting of rice may improve not only the physico-chemical properties of the earth but also the availability of macro and micronutrients in soil and zinc fertilization in Basmati rice may help in improving its grain quality.
Zinc deficiency in soils is prevalent worldwide, especially in high pH calcareous soils (Adriano, 2001;Fageria et al., 2003;Norman et al., 2003;Prasad, 2006;Alloway, 2008;Cakmak, 2002Cakmak, , 2008aCakmak, , 2008b. A recent analysis of two hundred forty-one thousand soil samples in India showed that 49% soils are deficient in Zn (Behera et al., 2009) and Zn deficiency is widespread in north Indian rice-wheat cropping system belt (Prasad, 2005). In rice, zinc deficiency is characterized by brown spots, which appear first in the younger leaves and later in the lower leaves. In severe Zn deficiency, burnt dark brown patches of plants in rice fields (Dobermann & Fairhurst, 2000). Zinc deficiency in rice was first reported by Nene (1966).
The response of rice to Zn has been reported by several workers in India (Srivastava et al., 2006;Shivay et al., , 2010Pooniya et al., 2012;Ghasal et al., 2018;Yadav et al., 2019), China (Shihua & Wenqiang, 2000) and the USA (Slaton et al., 2005). Zinc is now recognized as the fifth leading health risk factor in developing Asian countries, where rice is the staple food (Anonymous, 2007) and Zn nutrition of humans and animals has recently received considerable attention (WHO, 2002). Hotz and Brown (2004) estimated that 1.2 billion (20% of the world population) are at risk of inadequate Zn uptake. There is a HarvestPlus Global Challenge Programme of the Consultative Group on International Agricultural Research (CGIAR) focusing on breeding Zn efficient cultivars (HarvestPlus, 2009) to evolve rice and other cereals varieties with the denser-Zn grain. Nevertheless, there are problems in developing rice varieties with high grain yields and denser-Zn grains. However, as pointed out by Prasad (2009) this can be achieved at a faster rate through application of Zn fertilizers and that too without any compromise on grain yield under Zn stress conditions. Even when Zn-efficient cultivars are developed, adequate Zn fertilizers would be required to make up the Zn depletion of soils (Shivay et al., 2010;Prasad & Shivay, 2020).
Zn fertilizers (oxides, sulphates and other Zn salts) since, as with most crops, the normal way of correcting Zn deficiencies in soils is to apply these fertilizers (Brennan & Bolland, 2006;Alvarez et al., 2009). However, In India, Zn deficiency is usually corrected through the application of inorganic salt, mainly ZnSO 4 ·7H 2 O. Soil application of 5 kg Zn ha -1 (25 kg ZnSO 4 ·7H 2 O ha -1 ) is recommended for rice in India. However, the product is quite costly and generally not available to farmers', resulting in reduced rice yield. Other sources are the chelated forms of Zn such as Zn-EDTA, which supplies a substantial amount of Zn to the plants without interacting with soil components (Karak et al., 2005) because the central metal ion Zn 2+ is surrounded by chelate ligands (Mortvedt, 1979), needs to be investigated.
However, no reports are available on combining green manuring crops and Zn fertilization on productivity, Zn content and kernel quality of Basmati rice. Therefore, the current investigation was undertaken to quantify the combined effects of summer green manuring crops and zinc fertilization on productivity, Zn content and kernel quality of Basmati rice in summer green manuring-Basmati rice cropping system.

Description of the Study Area
The field experiments were conducted for two consecutive years at the ICAR-Indian Agricultural Research Institute, New Delhi, India during summer-Kharif/rainy seasons (April-October) of 2009 and 2010 on a sandy clay-loam soil (typic Ustochrept). The experiments in both the years were conducted with a fixed layout plan on the same site. The institute farm is located at a latitude of 28 o 38′ N, longitude of 77 o 10′ E and altitude of 228.6 m above the mean sea level. The mean annual rainfall of New Delhi is 650 mm, and more than 80% generally occurs during the south-west monsoon season (July-September) with mean yearly evaporation of 850 mm.

Application of Treatments and Fertilizers
During summer seasons three summer green manuring crops viz. Sesbania aculeata (Dhaincha), Crotalaria juncea (Sunhemp), and Vigna unguiculata (Cowpea) were planted as main plot treatments. A summer fallow treatment was also considered as the control. After 40 days the summer green manuring crops were incorporated into the soil before transplanting of rice. After incorporating green manuring crops, each main plot (with green manure incorporation or otherwise) was divided into six sub-plots, which received the Zn-fertilization treatments. The experimental field was disk-ploughed twice, puddled three times with a puddler in standing water and levelled. At final puddling 26 kg, P ha -1 as single superphosphate and 33 kg K ha -1 as muriate of potash was broadcasted. Addition of 26 kg P as single super phosphate also supplied 45 kg S ha -1 and took care of S deficiency (if any). It also takes care of the S advantage of ZnSO 4 ·7H 2 O and permits a fair comparison of ZnSO 4 ·7H 2 O and ZnO or EDTA-chelated Zn as a source of Zn. Nitrogen at 150 kg ha -1 as prilled urea was applied into two equal splits, half at the time of transplanting and remaining half at panicle initiation stage (40 DAT). In all the Zn treatments, uniformly 5 kg Zn ha -1 was applied at the time of transplanting.

Rice Transplanting
Three 25-day-old seedlings of Basmati rice (Oryza sativa L.) variety 'Pusa Basmati 1' was transplanted per hill at 20 cm × 10 cm in the first fortnight of July in both the years of study. It is a Basmati (aromatic) variety released from ICAR-Indian Agricultural Research Institute, New Delhi, India during 1989 for its commercial cultivation. It is a cross between 'Pusa 150' and 'Karnal Lokal'. It produces long slender grains with good aroma and excellent cooking qualities (Rani et al., 2009;V. P. Singh & A. K. Singh, 2009;Siddiq et al., 2012). Irrigation channels measuring 1 m wide were placed between the replications to ensure an easy and uninterrupted irrigation water flow. An individual plot was independently irrigated from the irrigation channels. Rice crop was grown as per recommended package of practices and was harvested in the second fortnight of October in both the years of experimentation.

Measurements of Yields of Rice
Harvesting of the Basmati rice was undertaken as soon as it attained the harvest maturity. The harvesting was done with the help of sickles after leaving the border area. Net plots were demarcated at first from the portion of the plot kept for recording grain yield. Plants from the demarcated net plot area were harvested, tied in bundles and taken to the threshing floor for drying and threshing. The harvested plants were dried for three days to bring down the moisture content to around 14%. After threshing, the seeds were cleaned, sun-dried and their weight was recorded. The yields in kg plot -1 were converted to tonnes ha -1 . The weight of the harvested plants after sun drying and before threshing was recorded. The straw yield was obtained by deducting the seed weight from the total weight. The grain and straw yields were expressed in tonnes ha -1 . The 1000-filled grains, taken at harvest, were first counted by a seed counter and then weighed to compute the 1000-grain weight.

Chemical Analysis of Zn Concentration in Rice Grain and Straw
At harvest, the dried plants were separated into grain and straw, ground in a milling machine, sieved through 0.7 mm sieve and analysed for Zn content separately. Dried plant samples were digested with di-acid [perchloric acid (HClO 4 ) + nitric acid (HNO 3 ) in 3:10 ratio] as per procedures described by Prasad et al. (2006). After digestion and extraction of samples, total Zn was estimated with the atomic absorption spectrophotometer (Perkin Elmer; Model-A. Analyst 100).

Milling Quality Parameter
Hulling (%): Well sun-dried paddy (rough or unhulled rice) samples of each treatment weighing 100 g from each replication were hulled in a mini "Satake Rice Mill" (Satake, 1990), the weight of brown rice was recorded, and the hulling percentage was calculated as: (1)

Milling (%):
To obtain uniformly polished grains, the hulled brown rice was passed through a 'Satake Rice Polishing Machine' (Satake, 1990) for 2 minutes. The polished rice was weighed, and milling percentage was worked out as under: Milling (%) = [Weight of milled rice (g)/Weight of rough rice (g)] × 100 (2) Head Rice Recovery: The milled rice was passed through an appropriate sieve to separate whole kernels from the broken ones. Head rice recovery (%) was computed as:

Kernel Cooking Quality Parameters
(1) Kernel Length and Breadth Before Cooking Ten milled kernels from each plot were taken at random and placed separately on a graph paper, and their length and breadth were measured using a 'Photo Enlarger' with a magnification of 3X. The actual mean kernel length and breadth was expressed in mm.
(2) Kernel Length and Breadth After Cooking Rice cooking technique was a simple modification of the technique used by Juliano and Perez (1984). A sample of ten kernels was taken in 15 cm long and 2.5 cm wide test tubes and pre-soaked in 5 ml of tap water for 30 minutes. The tubes were then placed in a water bath maintained at boiling temperature (using a Thermotech temperature controller TH-013; Thermotech, Gujarat, India) for 6-7 minutes. After cooking, the tubes were taken out and cooled under running water for 2 minutes. Cooked kernels were taken out of the tubes, and excess water was removed with a blotting paper. Length and breadth of cooked kernels were measured, as mentioned above. (

3) Kernel Elongation Ratio
Kernel Length Expansion Ratio: The kernel length expansion ratio was calculated by dividing the cooked kernel's length by its original length.
Length expansion ratio = L 2 /L 1 (4) where, L 1 and L 2 are kernel length before and after cooking, respectively.

Kernel Breadth Expansion Ratio:
The kernel breadth expansion ratio was calculated by dividing the cooked kernel's breadth by its original breadth.
Breadth expansion ratio = B 2 /B 1 where, B 1 and B 2 are grain length before and after cooking, respectively.

Protein Content
Protein content in Basmati rice grain was obtained by multiplying N concentration with a coefficient factor 5.95 (Juliano, 1979;Juliano, 1985). This factor is based on the nitrogen content (16.8%) of the major rice protein (Glutelin). The protein content was expressed in percentage.

Amylose Content
A sample of 1 g milled rice grains was gently crushed and made into fine powder in a vitreous pestle and mortar. The flour samples were stored to uniform moisture of 12%. One hundred mg sample was weighed carefully on an electric meter balance and transferred to a 100 milliliter (ml) volumetric flask. One ml of distilled ethanol was added and mixed well. Ten ml of freshly prepared 1 N NaOH solution was added to it. After gelatinization, the sample suspension was heated for 10 minutes in a boiling water bath. The volume was made up to 100 ml with distilled water. After thoroughly shaking the content, an aliquot of 2.5 ml was pipetted out into a 50 ml volumetric flask and added about 20 ml of water. Three drops of phenolphthalein indicator were added mixed well. The content was acidified by adding 0.1 N HCl drop by drop until the pink colour disappears. Then one ml of iodine reagent was added to develop blue colour and volume was made up to 50 ml. The absorbance at 590 nm was recorded with the help of a Spectrophotometer.
A standard curve was prepared based on the absorbance values of known quantities of pure amylose (rice amylose). The amount of amylose in the sample using the standard curve was prepared from pure amylose (range 0.2-1.0 mg) against a blank for which diluted 1 ml of iodine reagent to 50 ml with distilled water was used (Sadasivam & Manickam, 1992;Thanyumanavan & Sadasivam, 1984;Thimmiah, 1999).

Statistical Analysis
All the data obtained from rice crop for consecutive two years of study were statistically analyzed using the F-test as per the procedure given by K. A. Gomez and A. A. Gomez (1984). Least significant difference (LSD) values at P = 0.05 were used to determine the significance of differences between treatment means.

Grain and Straw Yields of Basmati Rice
In general, the grain and straw yields were higher during the second year of experimentation (Table 1). Basmati rice yields were significantly influenced by incorporating summer green manuring crop residues and Zn sources. The significantly higher grain and straw yields of Basmati rice were recorded when it was grown after Sesbania aculeata (Dhaincha) incorporation compared with Crotalaria juncea (Sunhemp), Vigna unguiculata (Cowpea) and summer fallow treatments.

Zn Concentration in Grain and Straw of Basmati Rice
Different green manures and Zn sources had significantly influenced on the Zn concentrations in grain and straw of Basmati rice (Table 1). In our studies, the Zn concentration in rice straw was higher compared with rice grains. The significantly higher Zn concentration in grain and straw of rice was recorded with Sesbania aculeata (Dhaincha) incorporation and application of EDTA-chelated Zn (12% Zn) during both the years. Zn concentration in grain and straw were significantly higher with Sesbania aculeata (Dhaincha) incorporation compared with Crotalaria juncea (Sunhemp), Vigna unguiculata (Cowpea) and summer fallow treatments.
In general, application of Zn fertilizers, irrespective of its source, significantly increased the Zn concentration in grain and straw compared with control (no Zn application). Among the Zn fertilization treatments, the application of EDTA-chelated Zn (12% Zn) resulted in statistically higher values of Zn content in grain and straw than all other Zn sources and control (no Zn application) during 2009 and 2010, respectively. Application of ZnSO 4 ·7H 2 O (21% Zn) was the second best treatment with respect to Zn concentration in grain and straw. The lowest values were recorded with control (no Zn application). A significantly higher correlation was recorded between grain yield and Zn uptake by grain ( Figures 1A and 1B). This might be because zinc affects carbohydrate metabolism through its effects on photosynthesis, sugar transformations and seed development. Thus, increased Zn content and its uptake in grains which helped to produce bolder grains, thus increasing the grain yield (Alloway, 2008).

Milling Quality Parameters of Basmati Rice
The data on hulling, milling and head rice recovery are presented in Table 2. Milling quality parameters of Basmati rice got significantly influenced by incorporating summer green manuring crop residue and Zn sources. In general, hulling, milling and head rice recovery percentages significantly improved with green manuring over summer fallows, irrespective of the type of green manure. The significantly higher values of milling quality parameters were recorded when it was grown after Sesbania aculeata (Dhaincha) incorporation compared with Crotalaria juncea (Sunhemp), Vigna unguiculata (Cowpea) and summer fallow treatments.

Kernel Length Before and After Cooking and Length Expansion Ratio
Incorporation of green manures and Zn sources had influenced significantly the kernel length before and after cooking (Table 3). Our results indicate that the rice kernels were significantly lengthier, both before and after cooking with Sesbania aculeata (Dhaincha) incorporation and application of EDTA-chelated Zn (12% Zn) during both the years. Kernel length before and after cooking and length expansion ratio were significantly higher with Sesbania aculeata (Dhaincha) incorporation compared with Crotalaria juncea (Sunhemp), Vigna unguiculata (Cowpea) and summer fallow treatments. Application of Zn fertilizers, in general, irrespective of its source, appreciably increased the kernel length before and after cooking compared with control (no Zn application). Among the Zn fertilization treatments, application of EDTA-chelated Zn (12% Zn) resulted in statistically higher kernel length values before and after cooking compared with all Zn fertilizer sources and control (no Zn application), respectively during 2009 and 2010. Application of ZnSO 4 ·7H 2 O (21% Zn) was second best treatment with respect to kernel length after EDTA-chelated Zn (12% Zn) however; it was statistically inferior to it. The lowest values were recorded with control (no Zn application). The kernel length expansion ratio was affected significantly with Zn sources during 2009 only. Length expansion ratio got increased with Zn application during 2010 also, but up to non-significant levels. However, it was highest with EDTA-chelated Zn (12% Zn) application.

Kernel Breadth Before and After Cooking and Breadth Expansion Ratio
Application of Zn and green manure incorporation significantly influenced the kernel breadth of Basmati rice both before and after cooking (Table 4). Kernel breadth increased significantly, both before and after cooking with Sesbania aculeata (Dhaincha) incorporation and application of EDTA-chelated Zn (12% Zn) during both the years. Kernel breadth before and after cooking was significantly higher with Sesbania aculeata (Dhaincha) incorporation compared with Vigna unguiculata (Cowpea) and summer fallow treatments.  Kernel breadth before and after cooking was significantly higher with Zn application, irrespective of its source, compared with control (no Zn application). Among the Zn fertilization treatments, application of EDTA-chelated Zn (12% Zn) resulted in statistically higher kernel breadth values before and after cooking compared with all other Zn fertilizer sources and control (no Zn application), respectively during 2009 and 2010. Application of ZnSO 4 ·7H 2 O (21% Zn) was the second best treatment with respect to kernel breadth increase. Application of ZnO (82% Zn) was least effective in affecting the kernel breadth of Basmati rice. The lowest values were recorded with control (no Zn application). However, incorporating green manures or Zn application could not significantly influence the kernel breadth expansion ratio during both the years.

Protein Content
Protein content in Basmati rice grain got significantly influenced by Zn application and green manure incorporation (Table 5). Significantly higher protein content in Basmati rice grain was observed when grown after Sesbania aculeata (Dhaincha) incorporation and application of EDTA-chelated Zn (12% Zn) during both the years. Protein content was significantly higher with Sesbania aculeata (Dhaincha) incorporation compared with Crotalaria juncea (Sunhemp), Vigna unguiculata (Cowpea) and summer fallow treatments.  Zn application, irrespective of its source, appreciably increased the grain protein compared with control (no Zn application). Among the Zn fertilization treatments, application of EDTA-chelated Zn (12% Zn) resulted in statistically higher grain protein values compared with all other Zn fertilizer sources and control (no Zn application), respectively during 2009 and 2010. Application of ZnSO 4 ·7H 2 O (21% Zn) was the second best treatment. The lowest values were recorded with control (no Zn application). Grain protein was 9.21 and 9.09% higher with EDTA-chelated Zn (12% Zn) application over control during 2009 and 2010, respectively. Application of ZnSO 4 ·7H 2 O (21% Zn) increased the grain protein content to the tune of 6.57 and 7.79% over control during 2009 and 2010, respectively. The performance of Zn sources in improvement in grain protein content was in the order; EDTA-chelated Zn (12% Zn) > ZnSO 4 ·7H 2 O (21% Zn) > ZnSO 4 ·H 2 O (33% Zn) > ZnSO 4 ·7H 2 O + ZnO (50% + 50%) > ZnO (82% Zn).

Amylose Content
Amylose content in Basmati rice grain got significantly influenced by Zn application and green manure incorporation (Table 5). The significantly higher amylose content values in Basmati rice grain were observed when it was grown after Sesbania aculeata (Dhaincha) incorporation and application of EDTA-chelated Zn (12% Zn) during both the years. It was significantly higher with Sesbania aculeata (Dhaincha) incorporation compared with Crotalaria juncea (Sunhemp), Vigna unguiculata (Cowpea) and summer fallow treatments.
Zn application, irrespective of its source, significantly improved the amylose content compared with control (no Zn application). Among the Zn fertilization treatments, application of EDTA-chelated Zn (12% Zn) resulted in statistically higher amylose content values than all other Zn fertilizer sources and control (no Zn application), respectively during 2009 and 2010. Application of ZnSO 4 ·7H 2 O (21% Zn) was the second best treatment. The lowest values were recorded with control (no Zn application) during both the years.

1,000-Grain Weight
Zn application and green manure incorporation significantly improved the 1,000-grain weight of Basmati rice (Table 5). Significantly higher values of 1,000-grain weight were recorded with Sesbania aculeata (Dhaincha) incorporation and EDTA-chelated Zn (12% Zn) application during both the years. It was significantly higher with Sesbania aculeata (Dhaincha) incorporation compared with Crotalaria juncea (Sunhemp), Vigna unguiculata (Cowpea) and summer fallow treatments.

Discussion
Zinc deficiency is a well-documented problem in food crops, causing decreased crop yields and nutritional quality. Generally, the regions in the world with Zn-deficient soils are also characterized by low crop productivity. Cereal crops, especially rice play an important role in satisfying daily calorie intake in the developing world. They are still inherently very low in Zn concentrations in grain, particularly when grown on Zn-deficient soils or under intensive cropping systems. Soil Zn deficiency is also a well-documented problem that reduces crop production. It brings about a significant decrease in plant performance, as shown in various countries such as in India, Pakistan, Australia, Turkey, USA and China (Alloway, 2008;Prasad, 2009;Prasad & Shivay, 2020). Earlier studies have shown that the inclusion of a green manure crop like Sesbania aculeata and other legumes in the rotation improves soil organic matter status and nutrient content and increases crop productivity (Yadvinder-Singh et al., 1991). However, the role of different types of Zn fertilizers, combined with the incorporation of green manure crops on grain quality parameters had not been undertaken so far.
This present study showed that there was a significant improvement in grain and straw yields, grain and straw Zn concentrations and kernel quality parameters of Basmati rice with summer green manure incorporation and Zn fertilization. This could be attributed to the higher supply of N and other micronutrient cations by incorporating leguminous green manures into the soil (Bisht et al., 2006;Pooniya & Shivay, 2011;Yadav et al., 2019). The increase in studied parameters of Basmati rice might also be due to higher nutrient availability and better physico-chemical soil properties under green manure incorporated plots. The increased availability of Fe and other micronutrients in soil with regular summer green manuring every year before transplanting rice in the rice-wheat system was responsible for higher yields in the green manuring plot than the non-green manuring plot (Nayyar & Chhibba, 2000). Sesbania aculeata (Dhaincha) supplied a significantly higher amount of readily decomposable organic materials (data not shown in this manuscript), which improved soil organic matter and nutrient status. It leads to recycling of nutrients into the soil and increased availability of nutrients and thus improved the yields and quality parameters of Basmati rice. Increase in the Zn concentrations in rice grains through soil Zn applications were positively stimulated by increasing soil N availability (Kutman et al., 2010). The positive effect of increasing N supply through leguminous green manure crops' residue incorporation on the Zn concentration of rice grain and straw was reflected in our studies. Nitrogen nutritional status of plants may also exert positive effects on Zn's root uptake . There are several steps during uptake and transport of Zn in plants which might be affected by N nutrition . By affecting root growth and stimulating root exudation of organic compounds (Marschner, 1995;Paterson et al., 2006), N may influence Zn's mobility and root uptake from soils . Nitrogen status in soil tended to affect the endosperm Zn concentrations to a greater extent than the whole grain Zn concentrations. The positive effects of high N on the endosperm Zn concentration have important implications for human nutrition. This part of the grain is the most commonly eaten part in many countries (Kutman et al., 2011). Enhanced nitrogen and other nutrients' supply through green manure residue incorporation resulted in significantly larger/bolder grains, improving the 1,000-grain weight (Table 5), and larger grains most probably have greater endosperm-to-whole grain ratios.
The positive effect of increasing N and other nutrients' supply through leguminous green manure crops' residue incorporation on rice grain protein content was reflected in our studies. This could be attributed to the higher supply of N and other micronutrient cations by incorporating legumes into the soil (Bisht et al., 2006;Pooniya & Shivay, 2011;Yadav et al., 2019). Grain protein concentration is an important quality parameter for rice, as it is the major staple food for the people in Asian and other developing countries.
production. They reported that out of various sources, the chelated-Zn (Zn-EDTA) was the most efficient Zn for low land rice production. These results are in the agreement with the findings of Karak et al. (2005) who reported that chelated Zn was the most efficient source of Zn for lowland rice production. Further, incorporating green manuring crops before transplanting of Basmati rice improves the organic matter content in soil. The applied Zn might have been complexed with the humic substances present in soil due to organic matter addition and there might have been lesser Zn fixation by the formation of insoluble Zn complexes. Thus, resulting into increase in the availability of soil applied Zn to rice plants. Improvement in the applied fertilisers' nutrient use efficiency by transplanted rice after green manure incorporation was also reported by Yadvinder-Singh et al. (1991). These results also confirmed those reported by Maftoun and Karimian (1989). Further, they concluded that Zn-EDTA's has greater influence over other sources of Zn in terms of growth and its utilisation by plants might be due to lesser retention and greater transport and movement of chelated Zn to plant roots. This could be attributed to lesser fixation in soil of Zn applied as Zn-EDTA than ZnSO 4 (Ortiz & Garcia, 1998). The higher increase in the Zn content in rice with EDTA-chelated Zn (12% Zn) might be due to increased amounts of Zn in soil solution that facilitates greater absorption of Zn as compared to other Zn sources. Application of ZnSO 4 ·7H 2 O (21% Zn) was second best treatment with respect to Zn concentration in grain and straw and its uptake after EDTA-chelated Zn (12% Zn). Singh et al. (1999) reported that applying the different sources of Zn up to 10 mg kg -1 increased the Zn concentration of rice leaves, being a higher uptake with Zn-EDTA than ZnSO 4 . Zinc content of rice grain and straw during the present investigation confirmed the results reported by Ugurluoglu and Kacar (1996) who studied the efficiency of ZnO, ZnSO 4 ·7H 2 O and Zn-EDTA on rice and reported that the application of Zn at the rate of 8 mg kg -1 as Zn-EDTA was found most effective in the enhancement of Zn content in rice plants. Rattan and Shukla (1991) studied the efficiency of Zn sources on rice cv. Pusa-33 in Typic Ustipsament and reported that the Zn content and uptake by rice were in the order of Zn-EDTA > Zn-DTPA > ZnSO 4 . Application of ZnO (82% Zn) was found to be least effective among all the Zn sources. This might be due to the fact that the EDTA-chelated Zn (12% Zn), ZnSO 4 ·7H 2 O (21% Zn) and ZnSO 4 ·H 2 O (33% Zn) are more water-soluble and therefore readily available, making its effects visible in the plants. In comparison, ZnO (82% Zn) is sparingly soluble and is not readily available. The water solubility of zinc sources is considered as an important criterion for Zn availability (Slaton et al., 2005a(Slaton et al., , 2005b. Mikkleson and Brandon (1975); Nayyar et al. (1990) also showed that ZnO was inferior to ZnSO 4 , both in grain yield and Zn uptake. Our results indicated that Zn applied through EDTA-chelated Zn (12% Zn) remained available to crops for a longer period of time than that with the rest of the Zn sources. This might be due to lesser transformation of applied Zn through EDTA-chelated Zn (12% Zn) into unavailable forms (Naik & Das, 2008).
The increase in rice's studied parameters with Zn fertilization may also be due to higher Zn availability to plants in treated plots than control (no Zn application). This might have lead to higher Zn uptake with Zn fertilization, resulting into higher biomass production (Shivay et al., 2008;Shivay & Prasad, 2012;Pooniya et al., 2012;Prasad & Shivay, 2018) and photosynthates translocation to reproductive parts (Ozkutlu et al., 2006;Alloway, 2008). A significantly high correlation was recorded between grain yield and Zn uptake by grain ( Figures 1A and  1B). This might be because zinc exerts an effect on carbohydrate metabolism through its effects on photosynthesis, sugar transformations and seed development. Thus, increased Zn content and its uptake in grains help in production of bolder grains, hence increasing the grain yield (Alloway, 2008). Our result showed that there was a significant improvement in grain and straw Zn concentrations with Zn application. High grain Zn concentration is considered a desirable quality factor that could increase the grains nutritional value for humans (Cakmak et al., 1998;Prasad, 2006;Shivay & Prasad, 2012;Prasad & Shivay, 2020). Also, the increased Zn concentration in rice straw is of immense importance from the viewpoint of cattle nutrition since in developing countries of Asia, rice straw is the major feed for farm cattle (Shivay et al., 2008). The positive correlations between Zn and protein content in grains of various cereal crops were observed by Cakmak et al. (2010). Strongly positive correlations between grain protein and Zn indicate that grain proteins represent a sink for Zn. Increase in protein content with Zn application in our results are in good agreement with the hypothesis that protein represents a sink for Zn in the grain (Morgounov et al., 2007;Cakmak et al., 2010;Kutman et al., 2010). The strength of the correlation between Zn and protein depends on sufficiently high Zn availability to the plants (Kutman et al., 2011). Application of Zn fertilizers in soil increased the Zn availability to the plants. Given that Zn plays a particular role in protein synthesis (Cakmak et al., 1989;Marschner, 1995;Kutman et al., 2011), enhancement in protein biosynthesis results from the increased N supply through leguminous green manures may also increase the sink strength for Zn. Ozturk et al. (2006) concluded that the highest accumulation of Zn in cereal grains occurs in the early stage of seed formation, the same stage during which the highest protein synthesis occurs (Kutman et al., 2011). In biological systems, proteins are highly dependent on Zn ions to maintain their activities. Zinc is needed for numerous proteins, having both a catalytic and a structural role (Anzellotti & Farrell, 2008). Further, the higher N supply by incorporating large amounts of plant biomass as leguminous green manures resulted in higher protein concentrations in rice grains, accompanied by higher Zn concentrations. In addition to N contribution, these summer legumes' biomass also recycled considerable quantities of P, K and other nutrients; and thus improving the rice grain quality parameters. Our results show the beneficial effect of summer green manure crops and Zn fertilizer application on grain protein content. The presence of protein, and likely of starch-protein interactions, affect the packing arrangement of starch polymers within the granule resulted in better kernel quality parameters. Also, zinc affects carbohydrate metabolism through its effects on photosynthesis, sugar transformations and seed/grain development (Alloway, 2008).  and Shivay and Prasad (2012) also reported the improvement in kernel quality parameters of rice with Zn fertilization. Thus, increased Zn content and its uptake in grains help in production of bolder grains, and hence improving the quality characters of Basmati rice. The present study also demonstrates that summer green manure incorporation and Zn fertilization improve the milling and cooking quality of Basmati rice. The increase in kernel length varied from 0.27 to 0.86 mm with green manuring and from 0.29 to 1.15 mm after cooking due to Zn fertilization, a quality for which Basmati rices are preferred and sold on a premium price all over the world for making rice Biryani (a dish made by cooking rice with mutton/chicken/peas and spices etc.).

Conclusions
Zinc fertilization and summer green manure incorporation increased the grain and straw yield, enhanced Zn concentrations and improved kernel quality in Basmati rice. The application of EDTA-chelated Zn (12% Zn) was the best in terms of grain and straw yield and Zn concentrations in grain and straw and kernel quality before and after cooking of Basmati rice. Application of ZnSO 4 ·7H 2 O (21% Zn) was second-best treatment followed by ZnSO 4 ·H 2 O (33% Zn) and ZnSO 4 ·7H 2 O + ZnO (50% + 50%). Application of ZnO (82% Zn) was least effective in affecting the studied parameters. The lowest values were observed with control (no Zn application). Among the summer green manuring crops, incorporation of Sesbania aculeata (Dhaincha) was found to be the best over Crotalaria juncea (Sunhemp), Vigna unguiculata (Cowpea) and summer fallow in terms of grain and straw yield, Zn concentrations in grain and straw and kernel quality before and after cooking in Basmati rice. Zn fertilization with EDTA-chelated Zn (12% Zn) lead to 25.91 and 21.26% higher grain yield; 60.66 and 82.14% Zn-denser grains; with 13.33 and 10.92% increase in head rice recovery in Basmati rice over control (no Zn application) during 2009 and 2010, respectively. The best results were obtained with Sesbania aculeata (Dhaincha) green manure among different green manures and EDTA-chelated Zn (12% Zn) among various Zn sources. Incorporation of summer green manures and adequate Zn fertilizer application in Basmati rice can thus lead to higher grain yield and Zn-denser grains with improved cooking quality in Basmati rice. Such positive effects of green manuring and Zn application can help sustaining good crop yields over time without deteriorating grain quality and nutritional value, especially in intensive cropping systems.