Screening of mulberry (Morus sp.) germplasm for quality traits

Introduction

Mulberry leaf quality is one of the key factors influencing the growth and development of silkworm, B. mori. The silkworm being a monophagous insect derives all the nutrients from mulberry leaf. Since the production of good quality cocoons depend on providing good quality leaves to silkworm, the development of superior quality leaf has become one of the prime objective in mulberry breeding programme. The leaf quality inturn is determined by the number of factors viz., mulberry variety, ecological factors, agronomical practices, method of harvest and preservation etc. Different quality traits such as leaf moisture content, proteins, carbohydrates, nitrogen, amino acids and chlorophyll are responsible for leaf quality (Bongale et al., 1991) [21] . Varieties possessing high nitrogen and amino acids contents in leaves are nutritively superior and promote growth and development of silkworm (Machii and Katagiri, 1991; Suryanarayanan and Shivashankar Murthy, 2002) [21,33] . According to some scientists, the combination of two traits i.e., nitrogen and total amino acid contents at higher quantity determines the leaf quality (Machii and Katagiri, 1991 and Suryanarayana and Sivashankar Murthy, 2002) [21,33] . Higher chlorophyll content in leaves indicates the photosynthetic efficiency; therefore it can be used as one of the criteria for quantifying photosynthetic rate in mulberry (Sujathamma and Dandin, 2000) [31] . More than 60 percent of the total cost of cocoon production is incurred for mulberry leaf production alone. Thus, improvement of mulberry for qualitative and quantitative trait is essential to sustain the profitability in sericulture. All the quality traits or nutrients may not be available at highest level in a genotype (Sujathamma and Dandin, 2000) [31] , as there are several factors, such as nutrients uptake, absorption of water, nitrogen utilization efficiency etc., which are involved and differ from accession to accession. Therefore, it is most essential to identify accessions possessing some of the important quality traits together, which may help to improve the growth and cocoon characters of silkworm. The effect of feeding of different mulberry varieties on larval growth, cocoon yield and other economic characters has been reported by number of investigators (Krishnaswami et al., 1970; Dar et al., 1988; Ninge Gowda and Sudhakar, 2002; Jalaja Kumar and Ram Rao, 2008) [17,10,23,15] . Since the production of fine quality cocoons depends on providing good quality leaves to silkworm, the development of superior quality leaf has become one of the prime objective in mulberry breeding programme. Appropriate selection of the tropical cultivars based on their effect on growth and cocoon yield parameters of B. mori races in different agro-climatic conditions is essential to select and exploit promising cultivars for better sericulture practices. The present investigation was undertaken to screen mulberry accessions for qualitative traits by analyzing important biochemical traits and rearing traits for fifteen mulberry accessions using ruling cultivated variety V1 as check variety.

Materials and Methods

The leaf quality characters of fifteen mulberry accessions viz., MI-0783, MI-0665, Mi-0065, MI-0789, MI-0669, ME-0095, ME-0174, MI-0532, MI-0632, MI-0497, MI-0828, MI-0845, MI-0657, ME-0174, MI-0300 belonging to different species were screened for quality traits through biochemical analysis and silkworm rearing. V1 mulberry variety was utilized as check variety in the present study.

Estimation of Biochemical Constituents

Leaf moisture content was estimated by following the method of Vijayan et al. (1997) [36] , The apical, middle and bottom leaves of several mulberry leaf samples were collected. After gently removing the water from the leaf surface with tissue paper, the collected leaf samples were cleaned under running tap water to get rid of dust and other residues. The fresh weight of each sample were recorded separately. Following a 24-hour drying period at ambient temperature and a 12-hour oven drying period at 60 °C, the dry leaf weight of each sample was promptly recorded. Using the following formula, the moisture content of the apical, middle, and bottom leaves were estimated and expressed as a percentage (%).

Fresh weight of leaves -Dry weight of leaves Fresh weight of leaves x 100

Total carbohydrate content was determined by phenol sulphuric method (Sadasivam and Manickam, 1996) [28] . Crude fibre and ash content were estimated by the method of AOAC (1955) [4] . The chlorophyll was estimated by following the procedure outlined by Hiscox and Israelstam (1979) [11] at the wave lengths of 645 nm and 663 nm using spectrophotometer. . The chlorophyll a, chlorophyll b and total chlorophyll content of leaf was computed using the formula suggested by Arnon (1949) [3] and expressed in mg/g of leaf tissue. Lowry's method (1959) was used to quantitatively measure the protein content.

Economic traits of silkworm, Bombyx mori L., Silkworms (B. mori L.) were reared by feeding leaves of individual mulberry accession during favorable commercial crops in three replications. To identify the accessions with superior leaf quality, rearing performance study was conducted with the silkworm double hybrid (CSR6 x CSR26) x (CSR2 x CSR27). Rearing traits viz., mature larval weight, effective rate of rearing, cocoon weight, shell weight, shell ratio were recorded. Silk quality parameters viz., silk productivity, silk filament length, filament weight, raw silk percent, denier and renditta were recorded. The experiment was repeated twice and average data on different leaf quality traits for biochemical constituents and economic traits of silkworm were statistically analyzed by completely randomized design (Cochran and Cox, 2000) [9] .

Results and Discussion

Biochemical Constituents 1. Moisture Content Significant variations were observed among the mulberry germplasm accessions with respect to moisture content (Table .1). Among the accessions, significantly high moisture content was recorded in MI-0669 (77.23%), whereas in V1 variety moisture content was 78.26 per cemt. The result of the present study is in conformity with the findings of Banerjee et al., (2007) [5] and Ahalya et al., (2020) [2] . Leaf moisture content is a genetic character and influenced by available soil moisture and root proliferation nature of mulberry variety. Feeding capacity of the silkworm larvae is improved by the presence of available moisture in the leaves, which accelerates their growth rate (Murthy et al., 2013) [22] . Most researchers noticed that, moisture content of mulberry leaves decreased gradually with corresponding increase in leaf growth. Thangamani and Vivekanandan (1984) [35] observed wide range of variation in eight varieties of mulberry for moisture content (63.67 to 70.60%) and total sugars (8.64 to 15.58%). Paul et al. (1992) [26] observed in their studies that availability of moisture content in leaves enhances the feeding efficiency of the larvae which inturn increases the growth rate.

Significantly maximum carbohydrate content was recorded in MI-0665 (22.10 g/100 g) followed by MI-0669 (21.86 g/100 g) while, V1 variety recorded 23.75 g/100 g. Current study results were enlightened by Ramamoorthy et al., (2018) [27] who reported highest protein content (24.8±0.22%) in the leaves of V1 variety. The V1 mulberry variety has highest level of total sugar (16.72%) and total protein (26.72%) when compared to the other five mulberry types, according to Kumar et al., (2009) [19] . Carbohydrate was reported to be chief source of energy for silkworm (Horie, 1978) [12] . Carbohydrates are estimated based on the amount of sugar and starch content available in leaves (Bose and Bindroo, 2001) [8] . If the leaves have high carbohydrate content, silkworm gain more energy and inturn may enhance the synthesis of silk protein.

Significantly higher crude fibre content was observed in leaves of MI-0669 (12.75%) followed by MI-0828 (12.54%) and it was 13.02 percent in V1 variety (Fable 1). Several workers have evaluated mulberry varieties and different species of mulberry for the crude fibre content. Salcedo et al. (2017) [29] reported that Morus nigra accessions showed higher crude fibre content (8.4 g/100 g) than Morus alba (7.1 g/100 g). Similarly, Iqbal et al. (2012) [13] reported highest crude fibre content in Morus nigra (12.3 g/100 g) and Morus alba (10.1 g/100 g). (Adeduntan and Oyerinde 2010) [1] reported crude fibre content in S36 (10.8%), Obeche (20.73%), S54 (13.70%) and in K2 (8.74%).

Total ash content was significantly maximum in leaves of MI-0669 (12.73%) followed by MI-0665 (11.87%) it was 13.06 percent in V1 variety. (Table 1). Iqbal et al. (2012) [13] studied that nutritive composition in the leaves of three indigenous species of mulberry namely, Morus alba, M. nigra and M. rubra and reported that the ash content varied between 8.91±0.51 percent to 11.73±1.09 percent.

Chlorophyll Content

Among the mulberry accessions, MI-0665 recorded the highest chlorophyll a (3.92 mg/g), chlorophyll b (1.87 mg/g) and total chlorophyll content (5.84 mg/g) (Table 1). The check variety V1 exhibited chlorophyll a, chlorophyll b and total chlorophyll content of 1.64, 1.95 abd 5.53 mg/g, respectively. Chlorophyll content is important for quantifying the photosynthetic efficiency of plant and is an essential constituent in assessing quality of foliage. Yadhav et al., (2022) reported that the highest total chlorophyll content was observed in S146 mulberry leaves. The silkworm fed with S146 mulberry leaves showed better cocoon performance effective rate of rearing (ERR%), cocoon weight, pupa weight, shell weight and filament length compared to other mulberry varieties.

Total Proteins

Among the fifteen mulberry accessions, MI-0669 (25.42%) recorded significantly maximum protein content followed by MI-0632 (24.77%) and V1 check variety recorded leaf protein content of 26.93 percent (Table 1). For many Lepidopteron larvae, the quality of the nutrients is mostly determined by the protein content in the leaves. It is well known that mulberry leaf protein directly biosynthesizes about 70 percent of the protein content of raw silk, specifically fibroin and sericin, with the remaining 30 percent coming from silkworm body tissue and hemolymph protein. This indicates the significance of leaf protein in the silkworm diet. Protein content present in different mulberry varieties had a direct influence on larval growth particularly in silk gland development and cocoon characters of silkworm (Shah et al., 2007 & Islam et al., 2023) [30,14] . Similar studies were conducted by Bongale and Chaluachari (1993) [6] observed a significant difference in protein content of eight different mulberry varieties leaves S54, C1, MR2, Kitchili, Roso, Kosen, Japanese and M5.

Economic traits of silkworm, Bombyx mori L., Economic traits of silkworm viz., mature larval weight (61.89 g/10 larvae) effective rate of rearing (92.44%), single cocoon weight (2.206 g), single shell weight (0.482), shell ratio (21.84%) silk filament length (1297 cm), silk productivity (6.881 g/day), silk filament weight (410 mg) and raw silk percent (18.59%) were significantly maximum when the leaves of MI-0669 accession were fed to the silkworm (Table 2), The variation in larval weight may be due to the difference in nutritional composition of the leaf. The present results are comparable with the findings of Tayade et al. (1988) [34] , who observed significant differences in weight of larvae when the silkworm larvae were fed on mulberry varieties viz., Kanav-2, Kosen, LM-1, LM-2, Mysore Local, S-30, S-36, S-41 and S-54. Patil et al. (2001) [25] reported that higher levels of leaf water content, total soluble sugars, soluble proteins, total chlorophyll and leaf water retention increased larval weight. Growth and development of silkworm (Bombyx mori L.) and the cocoon crop yield are influenced largely by the varietal difference and nutritional quality of mulberry (Morus sps.) leaf used as food (Parpiev, 1968 and Krishnaswami et al., 1970) [24,17] . Differences in silk quality parameters between the accessions might be due to difference in the nutritional contents in leaves of different accessions and larval duration during fifth instar. These results are in conformity with the findings of Sujathamma et al. (2001) [32] who reported significant differences all the characters viz., cocoon weight, shell weight, shell ratio, average filament length and filament denier, when the larvae were fed on different mulberry varieties. The food quality influences the weight of the cocoon, silk yield and physicochemical properties of silk thread. Kahn et al. (2007) evaluated seven mulberry genotypes through bio-assays and reported that the highest filament length was recorded in Ichinose (1206 m). Sujathamma and Bose et al. (1995) reported that succulent mulberry leaves with less fibre and high mineral content stimulate the metabolic activities in silkworm resulting in quantitative improvement of cocoon and silk.

Conclusion

Screening of fifteen mulberry accessions for quality traits revealed that MI-0669 and MI-0665 superior with respect to biochemical constituents and economic traits of silkworm. The silkworms fed on leaves of accession MI-0669 and MI-0665 were able to consume and digest more food which inturn led to increased larval growth. Hence, the silkworms which derived more nutrition from food produced good quality of cocoons with maximum cocoon and shell weights, shell ratio, productivity, filament length and denier. These findings provide a valuable foundation for selecting suitable genotypes for breeding mulberry varieties with enhanced nutritional profiles to optimize sericulture practices and increase silk yield.

Acknowledgement

AcknowledgmentsThe mulberry germplasm accessions used for the present study were procured from Central Sericultural Germplasm Resources Centre, Central Silk Board, Govt. of India, Hosur. The Authors thank CSGRC, Hosur, Tamil Nadu for providing the germplasm mulberry accession to the Department of Sericulture, Forest College and Research institute.

References

1. Adeduntan SA, Oyerinde AS. Evaluation of nutritional and antinutritional characteristics of obeche (Triplochition scleroxylon) and several mulberry (Morus alba) leaves. Afr J Biochem Res. 2010;4(7):175-178.
2. Ahalya BN, Chikkalingaiah M, Murali K. Evaluation of elite mulberry genotypes for growth and yield parameters in different seasons. J Entomol Zool Stud. 2020;8(4):1253-1256.
3. Arnon DI. Estimation of chlorophyll (DMSO). Plant Physiol. 1949;24:1-15.
4. Association of Official Agricultural Chemists (AOAC). Official methods of analysis. 8th ed. Washington (DC): AOAC; 1955. p. 118-119.
5. Banerjee R, Roychowdhuri S, Sau H, Das BK, Ghosh P, Saratchandra B. Genetic diversity and interrelationship among mulberry genotypes. J Genet Genomics. 2007;34(8):691-697.
6. Bongale UD, Chaluvachari G. Evaluation of four mulberry varieties by leaf biochemical analysis and bioassay with Bombyx mori L. J Indian Bot Soc. 1993;72:59-62.
7. Bongale UD, Chaluvachari G, Narahari Rao BV. Mulberry leaf quality evaluation of its importance. Indian Silk. 1991;39(8):51-53.
8. Bose PC, Bindro BB. A comparative biochemical study of seven promising mulberry (Morus alba L.) varieties under rainfed conditions of sub-tropical region. Indian J Seric. 2001;40:171-173.
9. Cochran WG, Cox GM. Experimental design procedures for the behavioural sciences. Cole Publishing Company; 2000. p. 319-380.
10. Dar HU, Singh TP, Das BC. Evaluation of mulberry varieties by feeding to Bombyx mori. Indian J Seric. 1988;27:16-22.
11. Hiscox JD, Israelstam GF. A method for the extraction of chlorophyll from leaf tissue without maceration. Can J Bot. 1979;57:1332-1334.
12. Horie Y. Quantitative requirement of nutrition for growth of the silkworm, Bombyx mori L. JARQ. 1978;12:211-217.
13. Iqbal S, Younas U, Chan KW, Sarfaraz RA, Uddin M. Proximate composition and antioxidant potential of leaves from three varieties of mulberry (Morus sp.): A comparative study. Int J Mol Sci. 2012;13(6):66516664.
14. Islam T, Bhat SA, Malik FA, Wani SA, Khan FA, Mir SA, et al. Feeding of different mulberry varieties and its impact on silk gland of silkworm, Bombyx mori L. Biol Forum Int J. 2023;15(1):488-492.
15. Jalaja Kumar S, Ram Rao DM. Characterization of seven mulberry genotypes for their leaf quality and bioassay with silkworm Bombyx mori L. Sericologia. 2008;48(1):85-93.
16. Khan IL, Malik GN, Dar HU, Baqual MF, Malik MA, Raja TA. Evaluation of some mulberry (Morus spp.) genotypes through chemo and bio-assay under temperate conditions of Kashmir. Indian J Seric. 2007;46(2):96-102.
17. Krishnaswami S, Noamani MKR, Ahsan M. Studies on the quality of mulberry leaves and silkworm cocoon crop production. Part 1. Quality difference due to varieties. Indian J Seric. 1970;9:1-10.
18. Kumar K, Mohan M, Tiwari N, Kumar S. Production potential and leaf quality evaluation of selected mulberry (Morus alba) clones. J Pharmacogn Phytochem. 2018;7(2):482-486.
19. Kumar L, Arunachalam G. Genetic variability in Morus alba L. by biochemical and bioassay methods for increased silk productivity. J Biomed Sci Res. 2009;1(1):11-18.
20. Lowry DH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951;193:265-275.
21. Machii H, Katagiri K. Varietal differences in nutritive values of mulberry leaves for rearing silkworm. JARQ. 1991;25:202-208.
22. Murthy VY, Ramesh HL, Lokesh G, Munirajappa DYB. Leaf quality evaluation of ten mulberry (Morus) germplasm varieties through phytochemical analysis. Int J Pharm Sci Rev Res. 2013;21(1):182-189.
23. Ninge Gowda KN, Sudhakar R. Evaluation of some exotic mulberry genotypes for leaf yield and quality. Bull Ind Acad Seri. 2002;6(2):39-49.
24. Parpiev BA. Water metabolism in silkworm fed with a different mulberry strain changing diet. Shelk. 1968;39:15-17.
25. Patil SV, Mallikarjunappa RS, Dandin SB, Eswara Rao MS. Evaluation of improved genotypes of mulberry through leaf chemical analysis and bioassay with silkworm race NB4D2. Sericologia. 2001;41(2):219226.
26. Paul D, Rao GS, Deb DC. Impact of dietary moisture on nutritional indices and growth of Bombyx mori L. and concomitant larval duration. J Insect Physiol. 1992;38(3):229-235.
27. Ramamoorthy R, Krishnakumar N, Paramanantham M. Comparative biochemical study of improved mulberry (Morus indica) cultivars. Int J Chem Stud. 2018;6(4):1211-1213.
28. Sadasivam S, Manickam A. Biochemical methods for agricultural sciences. New Delhi: New Age International (P) Ltd.; 1996.
29. Salcedo S, Amorós EM, Hernández AF, Martínez JJ. Physicochemical properties of white (Morus alba) and black (Morus nigra) mulberry leaves: A new food supplement. J Food Nutr Res. 2017;5(4):253-261.
30. Shah SIA, Khan IA, Hussain Z, Shah M, Usman A, Sadozai A. Studying the performance of silkworm, Bombyx mori L. races fed with different mulberry varieties. Sarhad J Agric. 2007;23(4):1079.
31. Sujathamma P, Dandin SB. Leaf quality evaluation of mulberry (Morus spp.) genotypes through chemical analysis. Indian J Seric. 2000;39(2):117-121.
32. Sujathamma P, Dandin SB, Savithri. Quality evaluation of mulberry (Morus spp.) genotypes through bioassay under Rayalaseema conditions of Andhra Pradesh. Indian J Seric. 2001;40(1):27-34.
33. Suryanarayanan N, Sivashankar Murthy TC. Differences in amino acid contents in leaf blades of mulberry (Morus spp.) varieties. Adv Plant Sci. 2002;15:475-481.
34. Tayade DS, Jawale MD, Unchegaonkar PK. Evaluation of some improved strains of mulberry by feeding experiment under Marathwada conditions. Indian J Seric. 1988;22(2):92-94.
35. Thangamani R, Vivekanandan M. Physiological studies and leaf nutrient analysis in the evaluation of best mulberry variety. Sericologia. 1984;24:317-324.
36. Vijayan K, Raghunath MK, Das KK, Tikader A, Chakraborthy SP, Roy BN, et al. Studies on leaf moisture of mulberry germplasm varieties. Indian J Seric. 1997;36(2):155-157.