Evaluation of sugarcane promising clones based on the morphophysiological traits developed from fuzz

Plant material and growth conditions

Sugarcane fuzz (true seed) of 78 crosses was acquired from Guangxi University, China. Sugarcane seed is tiny (approximately 1.8 mm × 0.8 mm) and tightly enclosed in bracts (Cheavegatti-Gianotto et al., 2011). In our experiments, seeds were not separated from the fuzz to mimic natural conditions, and the fuzz was not sterilized for the germination experiments.

Phenotypic and quality parameters data collection

This study evaluated 31 sugarcane genotypes including two local check varieties, CPF-251 and HSF- 240 (Table 1). A total of six traits, including five phenotypic cane traits such as plant height (PH), cane length (CL), tiller numbers (TN), internode length (IL), and cane diameter (CD), were measured using a ruler and vernier caliper at maturity stage (10–11 months old plants). One quality parameter of cane was measured by taking the Brix% (B) data at the maturity of the plant using the handheld refractometer.

Pigment analysis

For the measurement of Chlorophyll a and b method proposed by Arnon (1949) was used. Leaf samples with the same weight were immersed in ethanol overnight. Samples were gently shacked. With the help of a spectrophotometer (Cary 60 UV-Vis; Agilent, Santa Clara, CA, USA), the concentrations were recorded at different wavelengths, 663 and 645 nm (Adeel Zafar et al., 2021). The following equations were used for calculations: ${\displaystyle Concentrationofchlorophylla\left(\frac{mg}{g}F.W\right)=\left(12.7\ast A663\right)-\left(2.69\ast A645\right)}$ ${\displaystyle Concentrationofchlorophyllb\left(\frac{mg}{g}F.W\right)=\left(22.9\ast A645\right)-\left(4.68\ast A663\right)}$

Chlorophyll leaching assay

For the chlorophyll leaching assay, the method described by Qin et al. (2011), was utilized. The flag leaf was harvested from individual plants, cut into small pieces (about two cm), and immersed in 30 ml of 80% ethanol at room temperature with gentle shaking in the dark. Chlorophyll quantification was carried out in tubes at 0, 50-, 100-, 150-, and 200-min. Measurements were performed in a relatively dark room with feeble light.

Statistical analysis

The obtained phenotypic, quality, and physiological data of 29 sugarcane genotypes and two local check varieties for nine yield-related traits were subjected to descriptive statistical analysis using R software (Release 9.1.3; SAS Institute, Cary, NC, USA). Analysis of variance (ANOVA) was used to determine the variation in traits among the sugarcane genotypes (Aguiar et al., 2018).

The least significant difference (LSD) test-taking probability level was used to statistically separate the genotypes’ mean values (p <0.001). Using each trait’s mean sum of squares from ANOVA results, the following genetic parameters were estimated (Asante, Adjah & Annan-Afful, 2019).

Mean variability

Plant height (PH, cm)

Sugarcane’s higher plant height resulted from improved crop growing conditions and varietal characteristics. It significantly contributes to the expansion of crop biomass. In this study, plant height (PH) varied from 275.84 to 429.77 cm, with an overall mean of 383.98 cm (Table 3). The maximum PH was observed for CPF-251, while the smallest PH was recorded in NS-74 (Fig. 1A).

Table 3:

Summary statistics of 31 (including two local checks) sugarcane accessions for different agronomics traits.

Traits	Maximum	Minimum	Mean	Variance	Standard deviation
PH	429.77	275.84	383.98	1,499.58	38.72
CL	283.46	126.49	209.64	1,146.88	33.87
IL	17.25	8.75	12.87	6.58	2.56
TN	31.50	3.50	10.24	30.28	5.50
B	21.10	14.20	18.23	2.54	1.59
CD	36.88	21.27	27.66	10.74	3.28
Chl. a	14.12	2.64	5.97	6.75	2.60
Chl. b	9.99	1.64	4.84	3.42	1.85
T. Chl	24.11	4.44	10.81	19.20	4.38

DOI: 10.7717/peerj.15646/table-3

Notes:

PH

plant length (cm)

CL

Cane length (cm)

IL

Internodal length (cm)

TN

Tillers number

B

Brix (%)

CD

Cane diameter (mm)

Chl. a

Chlorophyll an (mg/g FW)

Chl. b

Chlorophyll b (mg/g FW)

T. Chl

Total chlorophyll contents (mg/g FW)

Tiller number (TN)

The yield of any crop depends on the number of productive tillers. The Tiller number (TN) varied from 3.50 to 31.50. Maximum TN was observed for the cultivar NS-6 (31.5), while less TN was observed in NS-54 (3.5). Overall, sugarcane genotypes showed more TN as compared to local checks (Fig. 2A).

Chlorophyll contents

Photosynthesis is a complex process that occurs in particular organs known as chloroplasts. The leaves acquire their green color from the direct absorption of blue and red sunlight by the green photosynthetic chlorophyll. In this study, NS-30 (14.12 mg/g FW) showed the highest amount of Chl a followed by NS-50 (10.51 mg/g FW), NS-72 (10.37 mg/g FW), and NS-75 (9.90 mg/g FW), while the genotypes NS-9 (2.63 mg/g FW), NS-5 (2.79 mg/g FW), and NS-8 (2.83 mg/g FW) showed less amount of Chl a (Fig. 3). We also observed Chl b in all the sugarcane genotypes. NS-30 (9.98 mg/g FW) showed the highest amount of Chl b, followed by NS-72 (7.99 mg/g FW) and NS-75 (7.64 mg/g FW). Furthermore, the genotypes NS-30 (24.11 mg/g FW) and NS-72 (18.36 mg/g FW) showed higher T. Chl, while the genotypes NS-5 (4.44 mg/g FW) and NS-6 (5.73 mg/g FW) have a minimum amount of T. Chl (Fig. 3).

Chlorophyll leaching assay

A chlorophyll leaching test was carried out to check the cuticular membrane permeability in all the mature leaves of 31 sugarcane genotypes (including two local checks). For this purpose, we checked the leaching at different time points (0, 50, 100, 150, and 200 min). A significantly higher amount of chlorophyll contents was extracted from the genotypes NS-30, NS-50, NS-63, NS-72, and NS-75 compared to the genotypes NS-5, NS-6, NS-9, NS-8, and NS-15 at the time point of 200 min (Fig. 4). An almost similar trend was observed for all the time points. These results showed a higher permeability rate in the earlier mentioned sugarcane genotypes, indicating the poor cuticle developments in the said genotypes.

Evaluation of genetic diversity through Principal Component Analysis

Principal component analysis (PCA) was previously used to estimate genetic diversity in different crops (Khan & Anwar, 2019). In PCA, a dataset is considered consultant, while the cumulative percentage of the variance of the essential additives is more than 80%. In this study, we also performed the PCA to check the variability and association relations of agronomic traits among the sugarcane genotypes (Fig. 5). Sugarcane genotypes were distributed in biplots by group, with traits represented by different vectors. The cumulative variance of the first eight PCs was 99.9%, and the variability among the PCs was 0.5 to 34.4% (Fig. 5A). The first three PCs (PC1 = 34.4%, PC2 = 22.5%, and PC3 = 13.1%) were significant. Chl a, Chl b, and total chlorophyll contents were the major contributing factors in PC1. Similarly, in the PC2 PH, CL, and IL were the main factors, while in the PC3, TN, TD, CL, and PH showed the highest loading values (Fig. 5B). Projection of traits showed that CL and IL strongly related to PH. Similarly, the traits Chl a, and Chl b are related to T. Chl (Fig. 5C). The genotypes NS-5, NS-9, NS-4a, and NS-15 were opposite to the NS-30, NS-50, NS-63, and NS-75 (Fig. 5C). Based on PCA results, these genotypes can be categorized as best and poor performers and can be utilized in future breeding programs.

Association among the morpho-physiological attributes of sugarcane

Association among any two traits is known as correlation. Pearson’s correlation results of this study showed that variation is present among the parameters. The traits such as PH, CL, and IL have strong associations. Similarly, PH showed a positive association with CL and IL, while it showed a negative association with the chlorophyll pigments. Chl a and Chl b have potent associations with T. Chl. Cane diameter negatively correlated with all the studied parameters (Fig. 6).

Cluster analysis of sugarcane accessions by using morpho- physiological attributes

The fuzz of exotic sugarcane crosses and two local checks (CPF-251 and HSF-240) were grown in the glasshouse and shifted to the field for further evaluation (Fig. 7). To study the diversity among the sugarcane accessions, hierarchical cluster analysis (HCA) was used by using recorded morphological data from the mentioned sugarcane genotypes. Such examination gives a superior circulation of accessions under various conditions as well as various gatherings of accessions in a similar climate. This method generated a phylogenetic tree based on the morpho-physiological attributes of sugarcane, on which the 31 sugarcane accessions including two local checks were distributed into four distinct clusters (similarity coefficient threshold = 25). These clusters were shown with different colors and named “tolerant (T, purple), moderate tolerant (MT, green), moderately susceptible (MS, red), and susceptible (S, blue). Additionally, these clusters were further divided into two distinct subgroups on the basis of similarities among the sugarcane accessions (Fig. 8).