Genetic Variability of Pigeonpea ( Cajanus cajan ( L . ) Millsp . ) for Water Logging and Salinity Tolerance under in vitro and in vivo Conditions

Pigeonpea (Cajanus cajan (L.) Millsp.) genotypes acquired from diverse sources, were screened for water logging and salinity tolerance under laboratory and field conditions. Analysis of variance revealed significant differences among the genotypes for various traits. Based on seedling vigor index, six genotypes (AL 1756, AL 1849, AH-06-7, H-2000-14, H-2003-14, ICP 5028) were found highly tolerant, 10 tolerant, 24 moderately tolerant, 12 sensitive and eight highly sensitive to water logging. In case of salinity treatment, seven genotypes (AL 1849, AH-06-7, H-2000-14, H-2001-25, Original Research Article Singh et al.; AJEA, 12(1): 1-13, 2016; Article no.AJEA.24071 2 H-2003-14, ICP 5028, JBP 110B) were found tolerant, 14 moderately tolerant, 17 sensitive and 22 highly sensitive. Mean values for germination percentage, seedling length, seedling dry weight and seedling vigor index were generally lower in salinity treatment than water logging treatment. Based on results of laboratory studies, 28 genotypes were screened in pot/field for tolerance to water logging and salinity and were categorized on the basis of plant survival percentage after treatments. Based on plant survival, four genotypes (AL 15, AL 1849, H-2000-14, H-02-28) were found highly tolerant, five tolerant, nine moderately tolerant, four sensitive and six were found highly sensitive in water logging treatment. In case of salinity treatment, six genotypes were found tolerant, five moderately tolerant, three sensitive and 14 were found highly sensitive. Based on higher plant survival (%) under both water logging and saline conditions, common genotypes were identified for their further use in breeding programme.


INTRODUCTION
Pigeonpea (Cajanus cajan (L.) Millsp.) is an important legume crop widely grown in many parts of Indian sub-continent.It is mainly used as human food, animal feed and an effective green manure crop.It is adapted to a wide range of environments and cropping systems.Major abiotic stresses encountered by pigeonpea are water logging, salinity and drought.These abiotic stresses adversely affect its productivity causing severe yield losses [1,2].Pigeonpea is reported to be highly sensitive to water logging [3,4] and salinity [5,6].Water logging and salinity stresses are important yield constraints in pigeonpea as water logging blocks oxygen supply to roots which hampers root permeability [7] and salinity impairs seed germination, reduces nodule formation, retards plant development and finally reduces crop yield [8].It is reported that germination and early vegetative growth stages in pigeonpea are more affected by water logging than the flowering stage [9].Water logging is reported to delay flowering, reduce vegetative growth, photosynthetic rate, biomass and grain yield [10,11].It has also been reported that short duration varieties of pigeonpea are more prone to the risk of yield reduction due to water logging as compared to medium or long duration varieties [12].Anoxic soil conditions are produced by excess water and consequently, plant roots suffer hypoxia or anoxia.Min and Bothalomew [13] reported gradual decrease in relative water content under water logging and plants wilt visually within a few hours of imposing water logging stress.It is observed that water logging induced decrease in leaf water potential [14] and membrane damage [15].Water logging also caused reduction in stomata conductance and plants exposed to water logging exhibit increased stomata resistance and limited water uptake leading to internal water deficit [16].Soil salinity is another major abiotic stress that affects plant growth, development and yield by causing physiological and biochemical changes in plants [17].It leads to osmotic stress and interferes with mineral nutrients uptake [18].Seedling and reproductive stages in plants are mainly affected by salinity stress [19].Salinity together with water logging can cause deleterious effects in plants posing major threat to crop productivity [20].These two abiotic stresses are related with each other as water logging results in rise of water table causing development of salinity in many parts of India [21].In India, saline water are defined as water having EC > 2 dS m −1 [22,23].A large genetic variation has been found in different cultivated and wild species of pigeonpea for salinity tolerance [5,24].In spite of the fact that both these stresses pose serious threat to pigeonpea production, very few efforts have been made to identify tolerant sources [4,11].Thus, it becomes important to identify the diverse sources of pigeonpea tolerant to water logging and salinity stresses to develop high yielding and tolerant cultivars keeping in view the present climatic conditions [25].Therefore, the present study was undertaken to screen and identify pigeonpea genotypes tolerant to water logging and salinity stresses under both in vitro and in vivo conditions.

Experimental Materials
The experimental material comprised a set of 60 pigeonpea genotypes, including advance breeding lines and some commercially released cultivars, acquired from different sources namely, International Crops Research Institute for Semi-Arid Tropics (ICRISAT), Patancheru; CCS Haryana Agricultural University, Hisar; Indian Agricultural Research Institute, New Delhi and Punjab Agricultural University, Ludhiana.

In vitro Screening
The 60 genotypes were evaluated under in vitro conditions in the laboratory of Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, India, using two treatments viz., water logging and salinity stresses during rainy seasons of 2012-13 and 2013-14.The water logging treatment comprised distilled water while the salinity treatment was having 30mM NaCl solution.Prior to the treatment, seeds of different genotypes were surface sterilized with 0.1% mercuric chloride for 1-2 minutes and residual chlorine was eliminated by rinsing the seeds with sterile distilled water several times.Seeds were submerged in beaker containing 200 ml normal distilled water in case of water logging treatment and in 200 ml 30 mM NaCl solution in case of salinity treatment, respectively, for 7 days (168 hrs) at 25±1°C.After the submergence treatment, seeds were then placed in sterilized petri-plates containing germination paper moistened with distilled water.For each genotype, 3 replications each were kept for both the treatments using 15 seeds per replication.Petri-plates were then kept in BOD incubator maintained at 25±1°C for 7 days.

Observations
Data were recorded after 7 days of incubation for both the treatments for germination percentage, seedling length (plumule and radicle), seedling dry weight (plumule and radicle) and seedling vigor index.Germination count was recorded by counting the number of germinated seeds out of the total seeds.Seed was considered to be germinated when emerging radicle elongated upto 1 mm length and then average germination percentage was computed.Seedling length was recorded by measuring the length of plumule and radicle and seedling dry weight was recorded after drying them in oven at 60°C for 3 days.Seedling vigor index was calculated using the formula: Seedling vigor index (VI) = mean germination% X mean seedling dry weight (mg).Based on seedling vigor index, the genotypes were categorized as highly tolerant (VI=>2000), tolerant (VI=1501-2000), moderately tolerant (VI=1001-1500), sensitive (VI=501-1000) and highly sensitive (VI=<500).

In vivo Screening
Based on results of laboratory screening, a set of 28 genotypes, including highly tolerant to highly sensitive, was chosen for in vivo screening in the pots/field.Plants of 28 genotypes were raised in 27 cm diameter plastic pots containing 10 kg of soil fertilized with recommended fertilizers as per package of practices for pigeonpea.Seeds of each genotype were sown in pots and replicated thrice for both water logging and salinity treatments.After germination, six plants were maintained finally for recording observations.After 30 days of sowing, the pots were kept for 5 days in ponds of 20 ft x 10 ft size containing normal water (water logging) and 30 mM NaCl solution (salinity).The water level was maintained at top surface level of pots.Five days after the water logging and salinity treatments, pots were removed from the ponds and placed on ground for the recovery of plants.During recovery of plants, normal water was applied as and when required.

Observations
Survival of plants was recorded after 5 days and then the final survival was recorded one month after water logging and salinity treatments.Chlorophyll content of plants on the third leaf from the top of plant was measured with SPAD (502-plus) before and 5 days after the water logging and salinity treatments.In each replication three plants were taken randomly for recording chlorophyll content.Percent reduction in chlorophyll content was calculated using the formula: Reduction in chlorophyll content (%) = (chlorophyll content before treatmentchlorophyll content after treatment)/chlorophyll content before treatment X 100.As suggested by Dua [26], the genotypes were categorized based on final plant survival percentage (PS), as highly tolerant (>90% PS), tolerant (75-90% PS), moderately tolerant (50-74% PS), sensitive (25-49% PS) and highly sensitive (<25% PS).Data on initiation and completion of leaf senescence were also recorded visually after water logging and salinity treatments.

Experimental Design and Statistical Analysis
Both the experiments (in vitro and in vivo) were conducted following Completely Randomized Design with three replications.The analysis of variance (ANOVA) was performed on single year data as well as on pooled data of two years using statistical analysis as per the procedure given by Snedecor and Cochran [27] and adapted by Cheema and Singh [28]

In vitro Screening
Analysis of variance, performed on single year data as well on pooled data of two years, revealed significant differences among the genotypes for all the traits studied viz., germination percentage, seedling length, seedling dry weight and seedling vigor index under both water logging and saline conditions (data not shown).In case of water logging conditions, six genotypes viz., AL 1756, AL 1849, AH-06-7, H-2000-14, H-2003-14 and ICP 5028 were found as highly tolerant, 10 genotypes (AL 15, AL 1758, AH-06-9, AH-06-12, H-2001-25, H-02-28, ICPL 332, ICPA 2039, ICPL 20128, JBP 110B) as tolerant, 24 as moderately tolerant, 12 as sensitive and eight as highly sensitive (Table 1).Under saline conditions, none of the genotypes was found to be highly tolerant, however, seven genotypes viz., AL 1849, AH-06-7, H-2000-14, H-2001-25, H-2003-14, ICP5028 and JBP 110B were found as tolerant, 14 as moderately tolerant, 17 as sensitive and 22 as highly sensitive (Table 1).Data on germination percentage, seedling length, seedling dry weight and seedling vigor index of 28 genotypes including some highly tolerant to highly sensitive genotypes are presented in Tables 2 and 3, respectively for water logging and saline conditions.It can be seen that genotype AH-06-7 had the maximum seedling vigor index (2655.0)followed by H-2000-14 (2636.3),AL 1756 (2099.8)and AL 1849 (2091.0)under water logging condition.The germination percentage of highly tolerant and tolerant genotypes was more than 75%.Out of the 60 genotypes, 3 highly sensitive genotypes recorded zero values for germination percentage, seedling length, seedling dry weight and seedling vigor index (data not shown).Under saline conditions, the genotype H-2000-14 had the maximum seedling vigor index (1841.9)followed by ICP 5028 (1706.3),AH-06-7 (1668.5)and H-2001-25 (1656.4).Germination of tolerant genotypes was more than 75%.Of the total 22 highly sensitive genotypes, five genotypes, having 0-15% germination, showed zero values for seedling length, seedling dry weight and seedling vigor index.However, some genotypes like AL 1760, ICPL 99051 and UPAS 120 recorded comparatively higher germination percentage (30-38.5%),but their seedling vigor index values (180.0-231.0)were very low due to low seedling dry weight as a result of less seedling growth after germination due to sensitivity to saline conditions.
Overall under in vitro condition, it was observed that mean values of all the genotypes for all the traits studied like germination percentage, seedling length, seedling dry weight and seedling vigor index were lower under saline conditions (63.1%, 6.4 cm, 15.4 mg, 1090.9, respectively) as compared to water logging conditions (72.8%, 7.7 cm, 19.1 mg, 1477.3,respectively).Overall there were 23 genotypes which showed similar reactions, i.e. tolerant (2 genotypes), moderately tolerant (5 genotypes), sensitive (8 genotypes) and highly sensitive (8 genotypes), under both water logging and salinity conditions.Of the six highly tolerant genotypes under water logging conditions, five genotypes namely AL 1849, AH-06-7, H-2000-14, H-2003-14 and ICP 5028 showed tolerant reaction, whereas one genotype, AL 1756 showed moderately tolerant reaction to saline conditions.Of the 10 tolerant genotypes under water logging conditions, two genotypes (H-2001-25, JBP 110B) showed the same reaction, i.e. tolerant, while seven genotypes namely AL 15, AL 1758, AH-06-9, AH-06-12, H-02-28, ICPA 2039 and ICPL 20128 changed their reaction to moderately tolerant, and one genotype, ICPL 332 changed its reaction to highly sensitive under saline conditions.Of the 24 genotypes that showed moderately tolerant reaction under water logging conditions, five genotypes namely AL 1779, AL 1839, AH-07-3, H-02-59 and SGBS 6 showed similar reaction (moderately tolerant), while the remaining 19 genotypes changed their reaction to either sensitive or highly sensitive under saline conditions.On the other hand, interestingly the genotype AH-09-9, which was found sensitive under water logging conditions, showed moderately tolerant reaction under saline conditions.

In vivo Screening
A set of 28 genotypes was also evaluated under field (in vivo) conditions for chlorophyll content and plant survival (%) under water logging and salinity conditions.Analysis of variance, performed on single year data as well on pooled data of two years, revealed significant differences for chlorophyll content before as well as after treatments, reduction in chlorophyll content after treatment and for plant survival percentage under both water logging and saline conditions (data not shown   In water logging treatment, the leaf senescence started on 4 th day from the lower part of plants and moved upwards and was nearly 100% after 10 th day of treatment in highly sensitive genotypes such as MAL 15, ICP 14085, ICPL99051 and ICPL 99050.In case of salinity treatment, the leaf senescence started on 2 nd day of the treatment in similar fashion as in case of water logging treatment and was nearly 100% after 6 th day of treatment in highly sensitive genotypes such as ICPL 14085, ICPL 99051, MAL 15, ICP 5028, ICPL 332, AH-06-9, AH-06-9 and ICPL 99050.In rest of the highly sensitive genotypes, complete leaf senescence was observed after 15-20 days of treatment.Comparatively lesser leaf senescence was observed in highly tolerant genotypes, while low to moderate leaf senescence was recorded in tolerant or moderately tolerant genotypes under both water logging and saline conditions.Chlorophyll content measured before the water logging and salinity treatments revealed that the genotype AH-09-9 had the maximum value (47.6) followed by H-2001-25 (47.3) and AH-06-9 (47.2).After water logging treatment, the maximum chlorophyll content was observed in AL 1849 (41.3) followed by AH-06-9 (40.5) and H-02-28 (39.3), whereas after salinity treatment, the maximum chlorophyll content was recorded by H-2001-25 (38.5) followed by AL 1849 (37.6) and AL 1756 (37.3).Overall, there were nine genotypes (highly tolerant or tolerant) under water logging conditions and six genotypes (tolerant) under saline conditions that showed <20% reduction in chlorophyll content (Tables 5,  6).On the average of all genotypes under in vivo conditions, it was observed that reduction in chlorophyll content was higher (36.19%) in saline conditions compared to water logging conditions (26.78%).
Results of the present study revealed that five genotypes namely AL 1756, AL 1849, H-2-28, AL 1758 and AL 15 recorded higher plant survival (>75%) under water logging as well as saline conditions, whereas six genotypes namely JBP 110B, ICPL 20128, MAL 15, ICP 14085, ICPL 99051 and ICPL 99050 were found highly sensitive under both conditions.The correlation coefficients between different traits, worked out for the 28 genotypes studied under in vivo conditions, are presented in Table 7. Plant survival percentage showed highly significant positive correlation with chlorophyll content both before and after water logging treatment.The plant survival also showed highly significant positive correlation with chlorophyll content after saline while before saline treatment the correlation between these two traits was nonsignificant.The reduction in chlorophyll content showed highly significant negative correlation with plant survival percentage and chlorophyll content both after the water logging and saline treatments.The reduction in chlorophyll content also showed significant negative correlation with chlorophyll content before water logging treatment, while after salinity treatment

DISCUSSION
The results of the present study indicated existence of large phenotypic variability among the genotypes screened in vitro under water logging and saline conditions.These results were in accordance with the results of previous studies [30][31][32].None of the genotypes was found to be highly tolerant under saline conditions, while six genotypes were found highly tolerant under water logging conditions.However, some of the genotypes showed similar reactions under water logging and saline conditions, but majority of the genotypes changed their reaction and showed lower level of tolerance to saline conditions as compared to their reaction under water logging conditions.Also the overall lower mean values for all the traits like germination percentage, seedling length, seedling dry weight and seedling vigor index under saline conditions as compared to water logging conditions.These results indicated that most of the pigeonpea genotypes used in the present study were more sensitive to saline conditions as compared to water logging conditions.Water logging results in anoxic conditions due to which respiration and electron transport are inhibited and ATP formation is decreased resulting in low seed viability and poor germination [33,34].Seed germination rate has been used as phenotypic marker for selection of salinity tolerant pigeonpea cultivars [35] where they observed genotypic variability to a greater extent under salt stress conditions.Earlier in chickpea, the parameters like germination percentage, radicle and plumule length and plant survival at maturity have been found effective in differentiating tolerant and sensitive genotypes under saline conditions [36].
The correlation coefficients between various traits indicated that genotypes having higher germination percentage will generally have higher seedling length, seedling dry weight and seedling vigor index.For instance the genotypes like AH-06-7 and H-2000-14 recorded higher germination percentage, also had higher seedling length, seedling dry weight and seedling vigor index, whereas genotypes like UPAS 120 and MAL 15 recorded lower germination percentage, also had lower seedling length, seedling dry weight and seedling vigor index.Seedling vigor index of seedlings was also found to be positively correlated with germination percentage, root length and seedling dry weight in previous studies [37,38].
In case of in vivo studies, significant differences were observed for chlorophyll content before as well as after treatments, reduction in chlorophyll content after treatment and for plant survival under both water logging and saline conditions indicated the existence of large genetic variability for both water logging and salinity tolerance in pigeonpea.Reduction in survival rate might be due to anoxia caused by water logging and salinity stresses.Overall, the lower mean values for chlorophyll content after salinity treatment and plant survival and high values for reduction in chlorophyll content indicated that saline conditions adversely affected the metabolism of plants which led to plant mortality.
The results revealed large extent of phenotypic variability in pigeonpea germplasm for tolerance to water logging and salinity stresses based on plant survival and reduction in chlorophyll content under field conditions.This variability can be exploited in breeding programme for developing high yielding cultivars having tolerance to water logging and salinity stress conditions [39].In chickpea also, plant survival was taken as an important parameter for identifying tolerant genotypes under salinity stress [26].Generally, it has been observed that water logging leads to low levels of oxygen in plants leading to internal water deficit.Oxygen deficiency leads to decline in net photosynthetic rate [40] which is due to reduced chlorophyll content and leaf area [41] and also due to stomata closure under stress [42].Water logging results in wilting, chlorosis, senescence and abscission of leaves which may be due to reduced uptake and mobilization of nutrients.Reduction in chlorophyll content was also reported in pigeonpea [43,44], wheat [45], maize [46] and Vigna sinensis [47].The highly significant negative correlation between reduction in chlorophyll content and plant survival indicated that high reduction in chlorophyll after water logging and saline treatments disrupted plant metabolism which led to plant mortality.
The genotype AL 1849 which was found highly tolerant or tolerant in both in vitro and in vivo condition under both water logging and saline conditions and the six genotypes namely H-2000-14, H-2003-14, H-02-28, AL 15, AL 1756 and AL 1758 which were found highly tolerant or tolerant in water logging and tolerant or moderately tolerant under saline conditions can be further evaluated under field conditions for their utility as cultivar as such or as donor for breeding for water logging and salinity tolerance.The genotypes which were found highly tolerant or tolerant and highly sensitive in both in vitro and in vivo conditions under water logging and saline conditions can be used for developing appropriate populations for genetic studies.Subarao et al. [6] studied comparative salinity tolerance among pigeonpea genotypes and their wild relatives.A large genetic variation was also observed earlier in cultivated and wild species of pigeonpea, especially C. scarabaeoides, for salinity tolerance [5].As the wild relatives of pigeonpea are rich reservoir of genes for various abiotic stresses, including water logging and salinity tolerance, they can be exploited for introgressing desirable genes into cultivated background to minimize the adverse effects caused by such stresses [48].It will help to broaden the narrow genetic base of pigeonpea which has always been a limiting factor in its breeding [37,49].

CONCLUSIONS
Results of the present study indicated that both water logging and saline conditions affect the germination under in vitro conditions and plant survival under in vivo conditions to varying extent suggesting that both stresses alone or together pose a serious threat to pigeonpea cultivation.Thus, a holistic approach towards the management of these stresses is needed by developing water logging and salinity tolerant cultivars.Some of the genotypes were found promising under both in vitro and in vivo conditions there by indicating correlation for seedling and adult plant tolerance which can be exploited in breeding programmes.

Table 4 . Correlation between various traits in pigeonpea genotypes under in vitro conditions
# WL: Water logging conditions; SAL: Saline conditions; **Significant at 1% level