Pod Trichome Characterisation Using Foldscope, Morphological Characterization and Genetic Diversity Among Indigenous Collections of Pigeonpea [Cajanus cajan (L.) Millsp.]

Present experiment was carried out during Kharif 2018 at Zonal Agriculture Research Station (ZARS), Kalaburagi, which is situated in agro-climatic zone-2 (North Eastern Dry Zone) of Karnataka state with 17^o 20' Latitude (N), 76^o 49' Longitude (E) and at an altitude of 443.88 meters above mean sea level.

 A total of 154 indigenous collections of pigeonpea obtained from National Bureau of Plant Genetic Resource (NBPGR)., New Delhi and three checks BSMR-736, Asha and PT-012 were sown in Augmented Block Design (ABD) consisting of 11 blocks with 14 genotypes in each block and checks were repeated randomly. Each genotype was sown in three rows of three meter length with spacing of 90 cm between rows and 20 cm between plants. Standard agronomic practices were followed and plant protection measures were taken as and when required by following the recommended package of practices (Anonymous., 2017). Observations were recorded on nine quantitative traits in five randomly selected plants from each genotype, viz., days to 50% flowering, days to maturity, plant height, number of primary branches, number of secondary branches, pod bearing length, number of pods per plant, seed yield per plant and 100 seed weight. Data recorded on five plants were averaged and average values were subjected to statistical analysis. The analysis of variance (ANOVA) was carried out for all characters individually. The data was analyzed using WINDOSTAT ver. 8.5 software developed by Indostat services, Hyderabad as per the principles of Mahalanobis (1936) and clustering by Tocher’s method (Rao, 1952) respectively.
 
Pod trichome length and density (per mm²)
 
Foldscope is an optical microscope that can be assembled from simple components, including a sheet of paper and a lens. It was developed by Dr. Manu Prakash and designed to cost less than US$1 to build. It provides magnification upto 140X. The kit includes magnets that can be stuck onto the foldscope to attach it to a smartphone, allowing the user to take pictures of the magnification. The prepared slide is inserted into the paper microscope and the pod trichomes were observed and counted for trichome length and trichome density respectively and pictures were taken with the help of smartphone.

The pod’s surface layer of test genotypes were cut into discs measuring 5 mm diameter and heated in 20 ml of water in glass test tubes on water bath at 85^oC. The water is then poured off and 20 ml of 96% ethyl alcohol was added and the contents were boiled for 20 min at 80^oC. Then alcohol was poured off and fresh alcohol was added until the chlorophyll content was completely removed. The sample was boiled at 85^oC by adding concentrated lactic acid until the pod segments were cleared. The test tubes were cooled, length of trichomes on pods was measured by gently pressing a sticky transparent tape on the pod surface, then fixed to a glass slide. The length of trichomes was observed and measured using the image J software. Number of trichomes per unit area on the epidermal layer of pod was counted under a foldscope. Mean data of five pods (3 observation from each pod) in each entry was used for analysis as trichome density.

Trichome length and trichome density, were correlated with shrivelled seed yield per plot due to pod fly incidence in 75 randomly selected genotypes.

Morphological characterization of 14 qualitative traits were recorded as per the key guidelines provided by PPV & FR (Protection of Plant Varieties and Farmer Rights) Authority, New Delhi and seed characteristics were recorded as per ICRISAT/ IBPGR (1993) (International Bureau of Plant Genetic Resources) guidelines.
 

Analysis of Variance (ANOVA) exhibited highly significant differences among genotypes for all the traits (Table 1). Based on D² values, the genotypes were grouped into 12 clusters using Tocher’s method given by Rao (1952). Of the 12 clusters, cluster I was the largest comprising 60 genotypes followed by cluster II (55 genotypes), cluster III (21 genotypes), cluster VII (11 genotypes), cluster X (3 genotypes) and seven clusters (IV, V, VI, VIII, IX, XI, XII) were solitary with single genotypes each. These results are similar to observations of Muniswamy et al., (2014) and Patel et al., (2018). Katiyar et al., (2004), observed 14 clusters while grouping of 221 genotypes whereas Nethravathi and Patil (2014) obtained nine clusters using 196 genotypes.

Generation of more clusters in general and sole clusters in specific is a representative of existence of huge amounts of diversity between the set of genotypes. The genotypes that fall into solitary cluster more usually have some distinctive characters which make them divergent. Furthermore, the genotypes which have congregated into a cluster exhibit narrow range of genetic diversity among them while, between clusters had broad range of variability. The generation of such clusters may be due to total isolation arresting the gene flow or rigorous natural or human selection for diverse adaptive complexes. The grouping of 154 germplasm lines into twelve clusters is presented in Table 2.



The highest intra cluster distance was noticed in cluster VII (55.57), followed by III (47.15), I (43.47), II (41.52) and X (37.95). The inter cluster D² values were maximum (241.23) between clusters V and IX indicating these two clusters are distantly placed, followed by clusters VI and IX (238.89), IV and IX (238.63). It is sensible to select genotypes from clusters showing high inter cluster distance for further crossing programme (Table 3). These results are in agreement with the earlier findings of Sreelakshmi et al., (2010), Muniswamy et al., (2014) and Patel et al., (2018).



Cluster mean analysis (Table 4) indicated that cluster IV (87.00) and II (92.45) are comprised of early flowering genotypes. Cluster XI (133.00) comprised early maturing genotypes followed by cluster VIII (136.00). The highest cluster mean for number of pods per plant was recorded in cluster IX (286.00) followed by cluster III (231.52). The highest cluster mean for seed yield per plant was recorded cluster IX (51.70) followed by cluster III (40.77). The maximum cluster mean for 100 seed weight was recorded in cluster XI (12.00) followed by cluster IV (11.50).  Similar results were also obtained by Muniswamy et al., (2014) and Patel et al., (2018).



Contribution of individual characters towards divergence have been calculated (Table 5), which revealed that the relative contribution of number of pods per plant has maximum (71.62%) followed by plant height (13.06%), number of days to maturity (8.93%), number of days to 50 per cent flowering (2.94%), seed yield per plant (2.90%) and rest of the genotypes have shown very negligible or no contribution towards diversity. While selection of genotypes, characters like number of pods per plant should receive maximum importance as they contributed more towards genetic divergence. These findings are in confirmative with previous results of Muniswamy et al., (2014), Singh et al., (2010), Bhadru (2011) and Hariprasad et al., (2018).



Trichome length and density
 
The average pod trichome length obtained was 0.22 mm per mm² and it ranged from 0.10mm (IC468576) to 0.39 mm (IC523145). The average pod trichome density recorded was 33.92 per 1 mm² and it ranged from 10.47 (IC468576) to 58.87 (IC523106). Estimation of phenotypic correlation coefficients for shrivelled seed yield per plot due to pod fly (Melanagromyza obtusa) incidence with pod trichomes in 75 randomly selected pigeonpea lines revealed negative non significant correlation indicating that the genotypes having high trichome length and trichome density suffered less damage by pod fly may due to antixenosis (Table 6). The genotypes IC523145 and IC553106 which had more trichome length and density respectively, had less shrivelled seed yield per plot (Plate 1, 2). These genotypes can be used in the breeding programme against pod fly host plant resistance. The above results and findings are in line with the findings of Moudgal et al., (2008), Revathi (2014) and Dhakla et al., (2010). The foldscope was used to measure rice root hair length and rice lemma trichome (Diwan and Kashappa, 2019).







Morphological characterisation
 
Out of 154 genoytypes, morphological variation was not observed for plant growth habit, leaf pubescence and pod pubescence. Unique genotypes were observed for characters like Dark purple stem colour (IC 523231), cylindrical pod shape (IC 468590), mottled, specked seeds (IC 468584) and Elongate seed shape (IC 523168). The traits like early plant vigour, branching pattern, base flower colour, stem colour, streak pattern of base petal, pod colour, seed characteristics exhibited lot of variation and the results of characterization of genotypes for morphological traits are presented in Table 7. Similar findings for plant vigor and plant growth habit were observed by Muniswamy et al., (2014) and Kumar et al., (2016). Hariprasad (2018) found similar results for branching pattern and stem colour. Kallihal et al., (2016) observed similar morphology in case of streaks pattern on base petal, pod shape and pod colour. Similar findings for seed colour pattern, base seed colour were found by Muniswamy et al., (2014).


 

Germplasm lines IC468574, IC523234, IC523226, IC523151, IC523126, IC468591 and IC523124 which fall into solitary cluster more often have some unique characters which make them divergent. Based mean performances of yield and yield related traits, the genotypes IC523144, IC523171, IC468593, IC523126, IC468590 were found promising. Hence, afore said genotypes can be used for the further studies for improving the yield as a parent in the future breeding programme. IC523145 and IC553106 genotypes which are having more trichome density and length can be used against the pod fly host plant resistance in plant breeding.