Assessment of drought tolerant cotton genotypes based on seedling & physiological attributes at different moisture levels

The potential of any crop variety is subjected to various environmental conditions and a large fraction of the potential yield cannot be attained. For this study, fifty cotton genotypes of different origin were collected from gene pool of Central Cotton Research Institute, Multan and screened in the glass house. These genotypes were also evaluated under the field condition to obtain parents that possess better tolerance against drought. Initial screening of these genotypes was conducted in the glass house under three moisture levels 100, 70 and 40% at field water capacity. Data were recorded for root length, fresh root weight, dry root weight, root shoot length ratio, shoot length; fresh shoot weight, dry shoot weight, fresh root shoot weight ratio and dry root shoot weight ratio in glass house. Five drought tolerant genotypes (BH-176, MPS11, DPL-45, Tree cotton and Cyto-62) and three susceptible genotypes (GS-444, Cooker-315, MS-64) were identified. Highly significant differences found among genotypes for all seedling characters. Further evaluation was done in field on the basis of various physiological parameters i.e. relative water content, excised leaf water loss, cell injury, leaf water potential, osmotic potential, net photosynthesis rate, transpiration rate and stomatal conductance under normal and water stress conditions. Same genotypes were confirmed under field studies. Highly significant genotypic variation existed among genotypes for all the physiological traits. The predicting genotypes may be selected and exploited to improve drought tolerance for the cotton crop.


Introduction
Cotton being a miraculous fibre that has a variety of uses, from fashion to home stuff to medical products.Cotton is noted for its versatility, appearance and performance.Cotton moved from field to fabrics as it provides thousands of useful products and supports millions of jobs.It is the key source of lint along with its by-products i.e. cotton seed oil, cake, hulls, and linters.In the international cotton set-up, Pakistan has the peculiarity in terms of cotton production and area.Cotton is the second most significant crop of our country after wheat, in terms of area, production and value added.Pakistan is the fifth largest producer of cotton all over the globe by producing 2170,000 million tons, the third leading exporter of raw cotton, the fourth major consumer of cotton (2467,000 million tons), and the prime exporter of cotton yarn [1].The potential of any crop variety is subjected to various environmental conditions and a large fraction of the potential yield cannot be attained.It is speculated that crop attains less than 25 % of the potential yield due to the adverse environmental conditions, and low water availability affected crop productivity nearly as much as all the other environmental factors combined [2,3].Amongst the abiotic stresses, deficiency of soil water constitutes a primary limitation to crop productivity in many regions of the world [4, 5].Water deficit is not only the cause of differences between actual yield and the potential yield, but it also results in yield instability of crops.Both surface and ground water resources in Pakistan are inadequate to meet the growing requirements for irrigation of agricultural crops.Drought has a very critical effect on growth, yield and quality characters [6].Limited water supply and high-energy costs can also decrease the yield of irrigated cotton.[7,8] have focused on root systems to enlarge water use efficiency.Root characteristics can be significant in predicting the response of plants to drought.Drought effect not only decreases plant height, shoot growth rate, and yield but also diminishes root growth.[9] have exhibited that root growth is less sensitive than shoot growth.Development of such genotypes which appears to be practicable, cost-effective and to tackle the problem of water shortage is an alternate way by genetic variation.It has been found from earlier studies that varieties/cultivars in each species vary from one another in their actions under drought conditions, signifying that drought tolerance in these groups can be improved through breeding.Physiological traits linked with drought tolerance in cotton have strong relationship with yield parameters.For example, photosynthetic rate; which significantly decreases with the imposition of water stress, can be effectively used for germplasm screening under drought conditions [10,11].Since, the response of germplasm with genetic variability may exhibit differential response under normal and water deficit conditions, regular screening of emerging germplasm need to be carried out for better adaptability and sustainable production.This study will help to understand the relationship of different seedling and physiological traits of cotton and their direct and indirect effects.The following studies were, therefore, to evaluate advanced cotton genotypes for drought tolerance characteristics under field conditions.

Screening of cotton genotypes in glass house
Initial screening was conducted in glasshouse under three moisture levels 100%, 70% and 40% at field water capacity.Seeds of all 50accessions were collected from the available stock at Central Cotton Research Institute, Multan.Seeds of accessions were planted during November, 2012 in polythene bags.Four hundred and fifty polythene bags with three repeats using completely randomized design were divided into three sets.One set was treated as control (T0) 100% and the other two as water stressed conditions 70% (T1) and 40% field capacity (T2).Seedlings grown under control condition with 100% water level (T0) were watered daily to keep the soil at field capacity.Water stress condition was developed by withholding water supply to the seedlings grown under water stress condition at two water level of 70% (T1) & 40% (T2), and the effect of water stress was monitored visually and with soil moisture meter (HH2 Theta Probe Type, Delta-T device, Cambridge, England).The data for each accession were recorded after 45 days of emergence.Plants were removed from the pots and gently washed to remove all sand.The plants were spread on paper for the measurement of fresh root length (g) and shoot length (cm).Plants were cut into roots and shoots, and fresh root weight (g) and fresh shoot weight (g) were calculated.Then roots and shoots dried for 72h at 60°C.Genotypes were evaluated for root length, fresh root weight, dry root weight, root shoot length ratio, shoot length, fresh shoot weight, dry shoot weight, fresh root weight, fresh shoot weight ratio and dry root shoot weight ratio.

Screening of cotton genotypes in field condition
Fifty cotton genotypes were screened under two irrigation levels i.e., well watered (T0) and water-limited (T1) in the field.The experimental design was Randomized Complete Block (RCBD) with three replications.Seeds of the genotypes were planted by maintaining 75 x 30cm row to row and plant to plant distance respectively.In order to avoid the rainfall effect, soil between the rows was covered with polythene sheet during the rainy period i.e.July-August as done by [12].Data of relative water content, excised leaf water loss, cell injury, leaf water potential, osmotic potential, net photosynthesis rate, transpiration rate and stomatal conductance were recorded.

Statistical analysis
The average data of all the seedling traits were subjected to statistical analysis.The least significance difference (LSD) was calculated at probability of 0.05%.Software packages of SPSS version 19 was used for data analysis [13].

Response of cotton genotypes in glass house
Variations were significant among all parameters for 50 genotypes (Table 1) under both stress levels suggested that root and shoot parameters as reliable traits for screening cotton germplasm for moisture stress.Genetic variation in 50 genotypes under drought and controlled conditions suggested the importance of root and shoot parameters in breeding programs [14,15].Highly significant differences were also noted in respect of genotypes (G), replication (R) as well as interaction of genotypes with replications (G × R).Stress produced a range of responses on the seedling traits measured.The presence of variability among genotypes for different traits under water stressed conditions has been reported [16,17].It is evident from the findings that the mean squares between all the accessions for all the seedling traits were highly significant under normal and moisture stress conditions (P≤ 0.01).

Root parameters
Results exhibited from (Table 2) that mean for all the root parameters under controlled were ranged from 6.77cm for (MS-64) to 13.97 cm for DPL-45.On the average basis DPL-45 had maximum root length (11.56cm) followed by BH-176 (11.42cm),Tree cotton (11.33cm) and MPS-11 (10.72cm).These genotypes performed better in all the conditions and these are considered as drought tolerant genotypes while entries GS-444 showed minimum root length following MS-64, Coker-315 and CIM-84 with root length of 4.8, 4.99, 5.4 and 5.6cm respectively.Due to drastic reduction in root length in stressed condition, these accessions may be rated as susceptible to water stress.
[18] reported in his findings that in cotton, root length was increased in mild and initialstage drought stress, but long-time reduced the root length as compared to control plants.On overall mean performance for fresh root weight, Tree cotton, F-14, GH-11-9-75 and MPS-11 had maximum mean values of 0.394, 0.387, 0.380 and 0.374g respectively while minimum fresh root weight was predicted by VS-212 (0.222g), Cooker-315 (0.224g), CIM-608 (0.226g) and GS-444 (0.230g).Root length-shoot length ratio under controlled and stressed conditions was maximum in F-14 followed by DPL-45, Cyto-62 and BH-176 with 0.910, 0.810, 0.794 and 0.778 respectively.These findings were very similar to [19].[20] Reported that accessions may be rated as susceptible due to reduction in root lengths in stressed condition.Minimum ratio was exhibited by CRIS-533 (0.404), Stoneville-603 (0.436), CIM-84 (0.444) and VS-212 (0.457).[21,22] also reported that root related parameters in cotton are directly associated with drought tolerance.

Shoot parameters
For drought tolerance, shoot length has been used as a selection parameter [23,24].Under 100% moisture levels (Table 3) shoot length ranged from 12.07cm (MS-64) to 19.67 cm (VS-212) similarly at 70% and 40% moisture levels its range was 9.6-15.8and 4.07-11.50cmrespectively.Data on mean performance of shoot length exhibited that maximum shoot length was found in MPS-11, Tree cotton, BH-176 and VS-212, with values of 15.0, 14.9 and 14.9cm respectively and these entries were less affected by moisture stress revealing that these are tolerant as compared to remaining entries.Minimum shoot length was shown by MS-64 (7.87cm), GS-444(8.0cm),F-14 (9.29cm) and Cooker-315 (9.66cm).[25] Exhibited that shoot length can be used as selection criteria for drought tolerance.On overall performance under normal and stress conditions, shoot weight mean values showed that Cyto-62 and BH-176 were the most tolerant entries showing better performance with maximum shoot weight of 1.91 and 1.82g respectively.The genotypes GS-444, L-229-29-71and Cooker-315 had minimum fresh shoot weight of 0.93, 0.97 and 1.03g respectively.In cotton, shoot dry weight is commonly used as a parameter for screening cotton genotypes against drought stress.Under normal condition; minimum dry shoot weight was exhibited by GS-444, Coker-315 and MS-64 with an average value of 0.28, 0.27 and 0.27g respectively.Minimum values for shoot weight at both stress levels were shown by the genotypes Cooker-315(0.19),MS-64(0.189) and GS-444(0.151g)respectively at 70% moisture level.While under 40% moisture level it was found as 0.109, 0.107, 0.103g and at mean of three levels it was observed as 0.192, 0.189, 0.178g respectively (Table 3).Based on root and shoot weight ratio; all accessions again appeared to respond differently both under normal and stressed conditions.On average based performance, under controlled and stress conditions maximum ratio was exhibited by the genotype L-229-29-71 followed by CRIS-9, F-14 and BP-52 with mean ratio of 0.91, 0.338, 0.327, 0.297 and 0.296 respectively.Minimum ratio was exhibited by NIAB-112, VS-212, CIM-84 and CIM-608 with values of 0.155 0.139, 0.145, 0.151 respectively.Entries performed better under stress condition exhibited low dry root dry shoot weight ratio as the stress level increased.Under 40% moisture level; entries i.e.Cooker-315 (0.322) and MS-64 (0.300) with poor performance under maximum stress showed maximum ratio while minimum ratio was observed for BH-176 followed by Cyto-62, DPL-45, MPS-11 and Tree cotton with ratios of 0.123, 0.132, 0.133, 0.134 and 0.141 respectively.It was concluded that diminution in performance for root and shoot parameters is due to the shortage of water supply and was also reported in the study of [20,26].

Response of cotton genotypes under field condition
Drought stress causes a wide range of adverse effects on physiological traits as well as productivity of cotton crop.Under drought conditions specifically in cotton leaves, net photosynthetic rate, transpiration rate, stomatal conductance, carboxylation efficiency and water potential decreased significantly [27].
The data obtained for all physiological traits under normal and stress conditions were subjected to pooled analysis of variance.Analysis of variance showed highly significant differences (P≤ 0.01) among cotton genotypes (Table 4).Genotype x environment interaction was also found highly significant for all the traits.

Relative water content (RWC).
In

Table 1 . Mean square for seedling traits of 50 Gossypium hirsutum L. accessions measured under control and water stressed conditions
Source of variation; Df, degree of freedom; SL, shoot length; RL, root length; SL/RL ratio, ratio of shoot to root length; RW, root weight; SW, shoot weight; SW/RW ratio, ratio between shoot and root weight; DRW/DSW ratio, dry root weight dry shoot weight ratio Cooker-315 (0.57).Maximum value was found in BH-176 (1.47), Cyto-62, Tree cotton, MPS-11 (1.20), and Stoneville (1.10)