sustainability

Article
Winter Tolerance Potential of Genetically Diverse Sugarcane
Clones under Subtropical Climate of Northern India
Ravinder Kumar 1, * , Mintu Ram Meena 1 , Pooja Dhansu 1, *, R. Karuppaiyan 2 , C. Appunu 2 ,
Neeraj Kulshreshtha 1 , Prashant Kaushik 3 and Bakshi Ram 2

1 ICAR Sugarcane Breeding Institute Regional Centre, Karnal 132001, India

2 ICAR Sugarcane Breeding Institute, Coimbatore 641007, India
3 Instituto de Conservación y Mejora de la Agrodiversidad Valenciana, Universitat Politècnica de València,
46022 Valencia, Spain
* Correspondence: [email protected] (R.K.); [email protected] (P.D.)

Abstract: The low temperature (LT) conditions that prevail during winter in subtropical regions
of India drastically affect the growth and yield of sugarcane. To identify low-temperature-tolerant
agronomical acceptable genotypes for immediate deployment as donor parents in the subtropical
sugarcane breeding program, 34 sugarcane clones belonging to 7 genetically diverse groups were
evaluated under three crop environments, viz., spring planting, winter ratoon and spring ratoon,
during 2015–2016 and 2016–2017. In the winter ratoon crop, commercial cane sugar and cane yield
were reduced, whereas sucrose % was increased over the spring planted crop and the spring ratoon
crop. The wild species and introgressed hybrid groups showed improvement for yield and quality
traits in the winter ratoon crop, whereas commercial and near commercial groups showed reduction
for these traits over the plant and spring ratoon crops. The tropical cultivars group was the poorest
performer irrespective of the traits and crops. Yield per se under a stress environment was adjudged
Citation: Kumar, R.; Meena, M.R.; as the best selection criteria. For classification of sugarcane clones according to their low temperature
Dhansu, P.; Karuppaiyan, R.; Appunu, tolerance, an index named winter tolerance index (WTI) is proposed which takes into account the
C.; Kulshreshtha, N.; Kaushik, P.; Ram,
winter sprouting index (WSI), winter growth and yield per se of the winter ratoon crop. The WTI had
B. Winter Tolerance Potential of
significant positive association with WSI, cane yield, millable cane population and cane length. As
Genetically Diverse Sugarcane
per the WTI ratings, the wild species of Saccharum complex and introgressed hybrid groups were
Clones under Subtropical Climate of
rated as excellent WT clones. Subtropical commercial or advanced generation groups were poor
Northern India. Sustainability 2022,
14, 11757. https://doi.org/10.3390/
WT clones, and tropical commercial cultivars group were winter sensitive clones. Clones such as
su141811757 AS04-635, AS04-1687, IK76-48, GU07-2276, IND00-1040, IND00-1038 and IND00-1039 had excellent
tolerance, and GU07-3849, AS04-245, Co 0238, AS04-2097 and GU07-3774 had good WTI scores. The
Academic Editor: Emanuele Radicetti
variety, Co 0238, may be continued for cultivation under LT regions with prophylactic measurers for
Received: 21 July 2022 red rot, while other clones listed above may be utilized in subtropical breeding programs.
Accepted: 7 September 2022
Published: 19 September 2022 Keywords: sugarcane; winter ratoon; spring ratoon; winter tolerance index; temperature stress
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. 1. Introduction
Sugarcane is an important cash crop, cultivated worldwide for industrial production
of sugar and bio-ethanol [1]. Being a C4 crop, it thrives well in both tropical and subtropical
regions of the world with a wide range of soils and temperature [2]. In a tropical climate,
Copyright: © 2022 by the authors. sugarcane passes through four distinct growth phases, viz., germination, tillering, grand
Licensee MDPI, Basel, Switzerland.
growth and maturity, whereas under the subtropical climate of Northern India, the crop
This article is an open access article
undergoes forced maturity at the onset of winter (during October to February) even if the
distributed under the terms and
crop was planted during summer (i.e., May planting after harvesting of wheat crop) [3]. The
conditions of the Creative Commons
optimum temperature for sugarcane growth is about 35 ◦ C, and temperatures below 20 ◦ C
Attribution (CC BY) license (https://
significantly limit its growth. At temperature below 12 ◦ C or 15 ◦ C, virtually no growth
creativecommons.org/licenses/by/
4.0/).
occurs [4–6]. In the subtropical states of India, every year, the prevailing low-temperature

Sustainability 2022, 14, 11757. https://doi.org/10.3390/su141811757 https://www.mdpi.com/journal/sustainability

Sustainability 2022, 14, 11757 2 of 15

(LT) stress (November to February) restricts the active cane growth period to 8–9 months
from the normal period of 10–12 months (in tropical states of India), resulting in low cane
yield and sugar recovery [7]. During LT stress, the sensitive cane varieties express several
physio-chemical changes, viz., reduction of water/mineral uptake and photosynthetic rate,
changes in membrane structure, disruption of ion homeostasis and accumulation of reactive
oxygen species (ROS) [8]. LT stress significantly reduces the cane yield and prevents or
delays bud sprouting and juice quality of sugarcane [9,10].
Temperature plays an essential role in synthesis and accumulation of sucrose in C4
plants such as sugarcane [11]. The production of phosphoenol pyruvate carboxykinase
is sensitive to low temperatures in C4 plants [12]. In the course of low-temperature
weather spells, juice acidity increases probably due to the formation of organic acids by
the conversion of stored sucrose in the stalk [13]. In subtropical India during winter,
below 10 ◦ C temperature distresses the activities of sucrose-synthesizing and hydrolyzing
enzymes, affecting the synthesis, movement and accumulation of sucrose, which results in
a drop in sucrose recovery due to low-temperature-induced inversion [14]. The quality of
sugarcane is principally articulated by the weather sequences met by the crop instead of its
absolute age [11,15] at harvest; however, the chance of age with calendar months may be of
some significance [16].
In the Northern India/subtropical region, autumn planted (October–November plant-
ing) and/or winter ratooned sugarcane crops (ratoon initiated during October–February)
undergo chilling (<14 ◦ C) and freezing stress (sub-zero) injury every year. In this region,
the minimum temperature during winter dips to 2–3 ◦ C, leading to very slow to no visible
growth in terms of germination, tillering, sprouting of ratoon crop and elongation of clumps
in most of the sugarcane varieties [17]. The farmers of subtropical states take only one
ratoon crop due to prevailing low temperature conditions at the time of harvesting of the
first ratoon crop during winter. Therefore, the ability to tolerate cold injury and the ability
to sprout immediately after the harvest of the ratoon crop during winter would determine
the success of sugarcane varieties in the subtropical region. Testing of cold or frost tolerance
potential of promising sugarcane varieties and clones are being commonly performed in
most of the countries that have low temperature conditions, viz., Australia, the United
States of America [18,19], South Africa [20], Brazil [21,22], China [8,23,24] and India [17].
In order to develop sugarcane varieties with better winter ratooning ability, it is
imperative to identify the potential donors with desirable traits to utilize in the sugarcane
improvement programs. At ICAR-Sugarcane Breeding Institute, Coimbatore, India, over
the years, several inter-specific and inter-generic hybrids have been developed, and their
evaluation under subtropical conditions was thought as a worthwhile exercise in identifying
valuable donors. Hence, the field study was planned to ascertain the degree of winter
tolerance potential among diverse sugarcane clones.

2. Materials and Methods

2.1. Experimental Plant Material and Layout
34 genetically diverse sugarcane clones including 8 subtropical early maturing cul-
tivars (STEM), 7 subtropical mid-late maturing cultivars (STMM), 3 tropical commercial
cultivars (TCC), 4 S. spontaneum introgressed hybrids (SSIH), 5 Erianthus arundinaceous
cyto-nuclear introgressed advanced generation hybrids (EAIAGH), 3 inter-specific hybrids
(ISH) derived from cold tolerant S. spontaneum collected from Arunachal Pradesh (GUC)
and 4 E. arundinaceus and S. spontaneum basic species clones (EASS) were planted in 6 repli-
cations during the spring season (spring planted crop) in randomized complete block
design during spring 2015–2016. Each clone was planted in two rows of six meter length at
an inter row space of 0.9 m. Two budded sets @ 12 buds/m were used for planting. The
recommended cultural practices for the region were followed to raise a good PC (plant
crop). Half of the experimental area comprising three replications was harvested in the
second fortnight of December 2015, while the remaining half was harvested during the first
fortnight of March 2016, and ratoon crops (RC) were allowed to initiate winter-initiated
 ing. The recommended cultural practices for the region were followed to raise a good PC
(plant crop). Half of the experimental area comprising three replications was harvested in
the second fortnight of December 2015, while the remaining half was harvested during
the first fortnight of March 2016, and ratoon crops (RC) were allowed to initiate winter-
Sustainability 2022, 14, 11757 3 of 15
initiated ratoon crop (WIRC) and spring initiated ratoon crop (SIRC). The ha−1 recom-
mended dose of major fertilizers, viz., N:P:K were applied @ 150N:50P:50K. The complete
dose of P and K wereratoonapplied as theand
crop (WIRC) based dose
spring at the
initiated timecrop
ratoon of planting of ha
(SIRC). The the
−1crop plant
recommended dose
or management ofofthe ratoon crop. A 1/4 dose of nitrogenous fertilizer was applied
major fertilizers, viz., N:P:K were applied @ 150N:50P:50K. The complete dose as of
a P and K
basal dose at the time of planting,
were applied as the whereas
based dosethe remaining
at the dose was
time of planting topcrop
of the dressed inmanagement
plant or three of
splits at 40–45 daystheafter planting
ratoon or ratooning
crop. A 1/4 (DAP/R),
dose of nitrogenous 60–75 was
fertilizer DAP/R andasata basal
applied the time
doseofat the time
earthing up (100–120of planting,
DAP) in whereas the remaining
the plant crop anddose wasIntop
SIRC. dressed
WIRC, theinremaining
three splits at
3/440–45
dosedays after
of nitrogenous fertilizer was top dressed in three splits at 75 days after rationing (DAR), DAP)
planting or ratooning (DAP/R), 60–75 DAP/R and at the time of earthing up (100–120
in the plant crop and SIRC. In WIRC, the remaining 3/4 dose of nitrogenous fertilizer was
120–135 DAR and at the time of earthing up (150–160 DAR). The dose of nitrogenous fer-
top dressed in three splits at 75 days after rationing (DAR), 120–135 DAR and at the time of
tilizer was kept 20% higher
earthing upfor the WIRC
(150–160 DAR).and
The SIRC.
dose of nitrogenous fertilizer was kept 20% higher for the
WIRC and SIRC.
2.2. Experimental Site, Topography and Climate
2.2. Experimental Site, Topography and Climate
The field experiment was conducted at the Research Farm of ICAR Sugarcane Breed-
The field experiment was conducted at the Research Farm of ICAR Sugarcane Breeding
ing Institute Regional Centre, Karnal, Haryana which is situated between 29.680 N lati-
Institute Regional Centre, Karnal, Haryana which is situated between 29.680 N latitude
tude and 76.990 Eand longitude at 243 m atabove
76.990 E longitude 243 mmean
above sea
meanlevel. The The
sea level. soilsoil
of the site
of the siteisiswell
well drained
drained alluvial sandy loam, having mildly alkaline (7.5–7.8) pH. The weather parameters
alluvial sandy loam, having mildly alkaline (7.5–7.8) pH. The weather parameters during
during the crop growth
the cropare givenare
growth ingiven
Figure 1 (2015–2016
in Figure and 2016–2017).
1 (2015–2016 and 2016–2017).

45
40
35
30
25
20
15
10
5
0
Min Max Min Max Min Max 2015 2015 2016 2016 2017 2017
2015 2016 2017 Min Max Min Max Min Max
Air Temperature Soil Temperature (10 cm)
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

300
250
200
150
100
50
0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Rainfall (mm) 2015 Rainfall (mm) 2016 Rainfall (mm) 2017

Figure 1. Weather data air and soil temp (◦ C) (min and max) and rainfall (mm) during 2015–2017.
Sustainability 2022, 14, 11757 4 of 15

2.3. Observations Recorded

In the PC trials, observations on metric traits, viz., germination percentage at 45 days
after planting (DAP), number of tillers per plot at 120 DAP, number of millable canes per
plot (NMC) at 240 DAP, stalk length, stalk diameter, single cane weight (SCW) and cane
yield at 10th month were recorded. Juice analysis was carried out using five randomly
selected stalks at the 10th month after planting; brix%, pol% and purity% of juice were
estimated in the laboratory as per ICUMSA methods [25].

2.4. Winter Tolerance Index (WTI)

To study the winter sprouting (WS) potential of the clones, in three replications stalks
of plant crops were cut at ground level during last week of December 2015 (coinciding
with peak winter). This block of three replications was reserved for taking observations in
the winter-initiated ratoon crop (WIRC), whereas the remaining three replications were
harvested during the first fortnight of March to take observations in the spring-initiated
ratoon crop (SIRC). The December ratooned stubbles were allowed to sprout during winter
(December to February). The number of stubbles (clumps) alive and sprouted per plot
and the number of shoots formed in each clump during winter were counted 45 days after
ratooning, coinciding with the onset of spring (February). The winter sprouting index
(WSI) was worked out following the formula suggested by Ram et al. [17] as follows:

% alive and sprouted clumps plot−1 × No of sprouts clump−1

Winter Sprouting Index =
100
To observe the active growth during winter, the stalks of unharvested replications were
marked at top visible dewlap during mid-November, and the winter elongation growth
(cm) was measured during the first week of March at the time of harvesting of the crop
for SIRC.
The WIRC and SIRC were raised during the 2016–2017 crop season by following
normal package and practice of cane cultivation of the region. Observations on metric
traits, viz., NMC per plot, stalk length, stalk diameter, SCW and cane yield were recorded
during November 2016. Juice analysis was carried out using five randomly selected stalks
in SIRC and WIRC trials at the 8th and the 10th month after ratooning (coinciding with
November) to record brix%, pol% and purity% in juice.
Based on cane yield of WIRC, WSI and plant elongation in winter months, an index
named the Winter Tolerance Index (WTI) is proposed as shown below:

WSI × plant elongation during winter × Cane Yield t ha−1

Winter tolerance index =
1000
On the basis of WTI, sugarcane genotypes were grouped into five categories as
shown below.
• WTI values: Tolerance category;
• >3.0: Excellent winter tolerance;
• 2.01–2.99: Good winter tolerance;
• 1.51–1.99: Average winter tolerance;
• 0.51–1.49: Poor winter tolerance;
• <0.50: Low temperature sensitive.

2.5. Statistical Analysis

The metric data were statistically analyzed using the SAS software version 9.3 (Strength-
ening Statistical Computing for NARS (iasri.res.in), and the means of different groups and
genotypes were compared by critical difference (C.D.) test at 5% level of significance.
Sustainability 2022, 14, 11757 5 of 15

3. Results
The mean sum of square (MSS) for crop nature (PC and WIRC; WIRC crop and SIRC),
genotypes and interaction (genotypes × ratooning season) were significant for all the
studied traits.

3.1. Comparison of Plant and Ratoon Crop Mean

The overall mean for CCS yield was significantly lower in WIRC (6.93 t ha−1 ) compared
to SIRC (7.83 t ha−1 ), and it was non-significant between PC (7.88 t ha−1 ) and SIRC. Lower
cane yield was recorded in WIRC (73.10 t ha−1 ) compared to PC (77.9 t ha−1 ), whereas the
cane yield of SIRC (81.7 t ha−1 ) was on par with that of PC. In addition to the differences
in cane and sugar yield between PC and WIRC, PC recorded higher millable canes, single
cane weight and cane length than ratoon crops (WIRC and SIRC). Although the cane yield
of WIRC was lower than the PC, the WIRC recorded significantly higher pol% (14.3%) than
plant crop (14.1%) (Table 1).

3.2. Comparison of WIRC and SIRC Mean

Sugar yield, cane yield and NMC recorded 11.49%, 10.53% and 6.58% improvement,
respectively, in SIRC over WIRC, whereas Pol% was 2.14% higher in WIRC (Table 1). The
higher pol% in juice in WIRC than SIRC might be due to an increase in crop age under
WIRC. The LT prevailed during the sprouting time of WIRC adversely affected the NMC
(an important yield contributing trait). As a result, the value of dependent traits, viz., cane
and CCS yield were reduced.

3.3. Inter Comparison of the Groups

The commercial clones, viz., subtropical early maturing cultivars (STEM) and subtrop-
ical mid-late maturing cultivars (STMM), produced significantly higher CCS yield (t ha−1 ),
pol% and SCW in PC than WIRC and SIRC over their respective experimental means
(Table 1). Similarly, these genetic groups (except STEM in WIRC) produced significantly
higher cane yield over the respective experimental means of the crop environments (PC,
WIRC and SIRC). However, tropical commercial cultivars (TCC) did not perform well
under the subtropical environment as indicated by their lower mean values for all the traits
under all crop types studied. E. arundinaceus cyto-nuclear genome introgressed advanced
generation hybrids (EAIAGH) were another important group which produced significantly
higher CCS yield (10.27 t ha−1 ), cane yield (95.00 t ha−1 ), pol (14.30% in PC and 14.80%
in WIRC), SCW (in PC 0.99 kg; SIRC 0.88 kg; WIRC 0.89 kg) and cane length (242.80 cm,
231.80 cm and 266.30 cm in PC, WIRC and SIRC, respectively) over the experimental mean,
but significantly lower for pol% than STEM and STMM groups. It is to be noted that most
of the clones belonging to the EAIAGH group did not perform well in WIRC and SIRC for
CCS and cane yield.
The number of millable canes was lower in the commercial (STEM, STMM, TCC) and
the near commercial (EAIAGH) group when compared to their respective experimental
means of test environments (PC, WIRC and SIRC). Three groups, viz., SSIH (S. spontaneum
introgressed immediate hybrids), EAIAGH and GUC (germplasm utilized clones) reported
significantly higher cane length (cm) over the experimental mean and all other groups in all
the three crop types. The cane yield of the SSIH and GUC group was significantly higher in
WIRC over the experimental mean, while the former group was on par (PC and SIRC), and
the latter group had either lower (PC) or higher (SIRC) performance over respective means
of the studied environment. The EASS (E. arundinaceus and S. spontaneum species clones)
group was the poor performer for CCS yield, cane yield, pol% and SCW in all the crop type
studied (PC, WIRC and SIRC) but was the best performer for NMC (Table 1).
Sustainability 2022, 14, 11757 6 of 15

Table 1. Performance of different categories of sugarcane clones for cane yield and juice quality traits in plant and ratoon crops.

Traits
Genetic CCS (t ha−1 ) Cane Yield (t ha−1 ) NMC (0 000/ha) Pol% 10 M SCW (kg) Cane Length (cm)
Group
PC WIRC SIRC PC WIRC SIRC PC WIRC SIRC PC WIRC SIRC PC WIRC SIRC PC WIRC SIRC
STEM 11.76 * 10.05 * 10.91 * 92.8 * 75.9 86.6 * 89.2 76.8 83.5 18.1 * 18.8 * 18.5 * 1.04 * 0.99 * 1.04 * 231.0 223.7 218.5
STMM 9.7 * 9.3 * 10.45 * 81.2 79.8 * 91.4 * 83.0 80.7 85.7 17.2 * 17.0 * 16.5 * 0.98 * 0.99 * 1.07 * 206.9 195.7 196.2
TCC 7.44 3.49 5.84 66.5 30.1 52.5 75.7 47.1 69.4 15.9 * 16.6 * 16.2 * 0.88 * 0.64 0.76 * 209.4 185.8 178.3
SSIH 4.27 4.85 4.28 79.5 93.7 * 80.5 125.8 * 146.6 * 131.4 * 8.7 8.7 8.8 0.63 0.64 0.61 285.5 * 271.7 * 261.2 *
EAIAGH 10.27 * 6.94 7.64 95.0 * 66.5 76.1 95.5 75.4 85.8 14.3 * 13.9 14.8 * 0.99 * 0.88 * 0.89 * 242.8 * 231.8 * 226.3 *
GUC 3.49 5.03 7.81 67.8 89.1 * 91.8 * 126.3 * 163.6 * 208.2 * 9.4 11.1 10.1 0.54 0.54 0.44 238.9 * 237.2 * 233.3 *
EASS 2.18 3.55 2.37 35.3 63.3 60.5 140.2 * 283.0 * 281.9 * 8.1 7.5 6.8 0.25 0.22 0.21 193.8 207.9 198.7
Mean 7.88 6.93 7.83 77.9 73.1 81.7 101.2 114.9 123.0 14.1 14.3 14.0 0.76 0.70 0.72 229.7 222.0 216.1
CD 5%
Season 0.54 6.4 4.29 0.14 0.015 4.29
Group 0.4 4.66 5.56 0.2 0.022 6.56
Interaction 0.69 8.08 11.36 0.4 0.039 11.36
Note: CCS = commercial cane sugar; NMC = number of millable cane; SCW = single cane weight; PC = plant crop; WIRC = winter-initiated ratoon crop; SIRC = spring initiated ratoon
crop; STEM = subtropical early maturing clones; STMM = subtropical mid late maturing clones; TCC = tropical commercial clones; SSIH = Saccharum sponteneum introgressed hybrids;
EAIAGH = Erianthus arundinaceus cyto-nuclear introgressed advanced generation hybrids; GUC = germplasm utilized clones; EASS = Erianthus arundinaceus S. spontaneum. * indicates
significantly superior over experimental mean.
Sustainability 2022, 14, 11757 7 of 15

3.4. Inter-Crop Environmental Comparison of the Groups

3.4.1. Subtropical Early Maturing Commercial Cultivars (STEM)
Commercial type early varieties recorded significantly higher sugar and cane yield
and NMC population in the plant crop than the ratoon crops, and between ratoons, it was
higher in SIRC than WIRC (Table 1). The performance trend for pol% was the reverse,
where WIRC (18.80%) had higher performance over SIRC (18.5%) and PC (18.10%). The
SCW was on par between PC (1.04 kg) and SIRC (1.04 kg), and their performance was
higher over WIRC (0.99 kg). The cane length was significantly higher in PC (231.00 cm)
and WIRC (223.70 cm) and had on par performance with SIRC (218.50 cm).

3.4.2. Subtropical Mid-late Maturing Commercial Cultivars (STMM)

The group had clear-cut differences for the expression of CCS t ha−1 in different crop
environments. The highest CCS yield was exhibited by SIRC (10.45 t ha−1 ), followed by
PC (9.70 t ha−1 ) and WIRC (9.30 t ha−1 ). The cane yield of the group was the highest in
SIRC (91.40 t ha−1 ) over PC (81.20 t ha−1 ) and WIRC (79.80 t ha−1 ). The latter two growth
environments had insignificant differences. The pol% in descending order was PC (17.20%),
followed by WIRC (17.00%) and SIRC (16.50%). The heaviest SCW was attained by the
group in SIRC (1.07 kg), followed by WIRC (0.99 kg) and PC (0.98 kg). The trend for cane
length in descending order was PC (206.9 cm), followed by WIRC (195.6 cm) and SIRC
(196.2 cm).

3.4.3. Tropical Commercial Cultivars (TCC)

The group had clear differences for the important traits in all three crops. The perfor-
mance for cane yield and its contributing traits was very high in PC over SIRC and WIRC.
The WIRC had extremely low performance for CCS yield (113.2% less over PC; 67.3% less
over SIRC), cane yield (120.9% less over PC; 74.4% less over SIRC), NMC (60.7% less over
PC; 47.35% less over SIRC) and SCW (37.5% less over PC; 18.75% less over SIRC).

3.4.4. S. spontaneum Introgressed Immediate Hybrids (SSIH)

The group had better performance under WIRC for CCS (t/ha), cane yield and NMC
population over PC, with an improvement of 13.58%, 17.86% and 16.53%, respectively.
There were no significant differences among the crop environments for SCW and pol%.
Similarly, the differences between PC and SIRC were insignificant for most of the studied
traits. This indicates that the group has better winter tolerance potential.

3.4.5. E. arundinaceus Cyto-Nulcear Genome Introgressed Advanced Generation

Hybrids (EAIAGH)
The best performance of the group for CCS yield (10.27 t ha−1), cane yield (95.00 t ha−1),
NMC (95.50 000/ha), SCW (0.99 kg) and cane length (242.80 cm) was observed in PC,
whereas the worst performance was recorded in WIRC with a reduction of 47.98%, 42.86%,
16.53% and 4.70% for CCS yield, cane yield, NMC and cane length, respectively. The
pol% values were significantly higher in SIRC (14.80%) compared to PC (14.30%) and
WIRC (13.90%).

3.4.6. Germplasm Utilized Clones (GUC)

The performance for CCS yield, cane yield and NMC, in descending order, was in SIRC
(CCS yield: 7.81 t ha−1 ; cane yield: 91.80 t ha−1 and NMC: 208.20 thousands/ha) followed
by WIRC (CCS yield: 5.03 t ha−1 ; cane yield: 89.1 t ha−1 and NMC 163.6 thousands/ha) and
PC (3.49 t ha−1 CCS yield; 67.8 t ha−1 cane yield and 126.3 thousands/ha NMC). Pol% was
the highest in WIRC (11.1) followed by SIRC (10.1) and PC (9.4). The better performance of
ratoon crops (WIRC and SIRC) over PC for cane yield and juice quality traits.
Sustainability 2022, 14, 11757 8 of 15

3.4.7. E. arundinaceus and S. spontaneum Species Clones (EASS)

The WIRC produced significantly higher CCS yield (3.55 t ha−1), cane yield (63.30 t ha−1),
NMC population (283.00 thousands/ha) and cane length (207.90 cm) over PC. The highest
improvement was observed in the NMC population in WIRC (101.85%) and SIRC (101.07%)
compared to PC.

3.5. Identification of Traits for Winter Tolerance

The winter tolerance index had strong and positive association with WSI (0.62 **),
cane yield (0.49 **), NMC (0.57 **) and cane length (0.46 **), whereas its association with
CCS yield was insignificant (Table 2). On the contrary, single cane weight (−0.35 *), cane
diameter (−0.45 **) and pol% −0.57 **) had negative association with WTI. The association
of WSI was significant and positive with NMC (0.80 **), whereas it was insignificant
with CCS yield, cane yield and cane length. WSI showed negative association with SCW
(−0.63 **), cane diameter (−0.71 ** and pol% (−0.76 **).

Table 2. Pearson Correlation coefficients between Winter tolerance index, Winter sprouting index,
yield and quality parameters.

WTI WSI CCS t ha−1 Cane Yield NMC SCW Cane Dia Cane Length pol%
WTI 1
WSI 0.62 ** 1
CCS t ha−1 −0.10 NS −0.30 NS 1
Cane yield 0.49 ** 0.20 NS 0.65 ** 1
NMC 0.57 ** 0.80 ** −0.28 NS 0.09 NS 1
SCW −0.35 * −0.63 ** 0.65 ** 0.37 * −0.76 ** 1
Cane Dia −0.45 ** −0.71 ** 0.55 ** 0.17 NS −0.84 ** 0.91 ** 1
Cane length 0.46 ** 0.22 NS 0.19 NS 0.70 ** 0.03 NS 0.27 NS 0.09 NS 1
pol% −0.57 ** −0.76 ** 0.52 ** −0.12 NS −0.69 ** 0.62 ** 0.76 ** −0.37 * 1
Note: WTI = winter tolerance index; WSI = winter sprouting index; * and ** refers to significant at p = 0.01
and 0.001.

The correlation of other variables indicates that the cane yield had strong and positive
association with CCS yield (0.65 **) and cane length (0.69 **), whereas its association with
NMC, cane diameter and pol% were non-significant. Pol% had positive association with
CCS t ha−1 (0.52 **), SCW (0.62 **) and cane diameter (0.75 **), whereas with NMC (−0.69 **)
and cane length (−0.36 *), it had negative association.

3.6. Classification of Groups Based on Winter Tolerance and Winter Sprouting Index
The WTI and WSI scores of SSIH, EASS and GUC groups indicate the excellent winter
tolerance and winter sprouting potential of these groups (Table 3). When comparing both
the indexes, WTI indicates the superiority of SSIH (5.66) group over EASS (3.71) and GUC
(3.00), whereas per WSI, the group EASS (5.05) was the best performer followed by SSIH
(4.09) and GUC (3.96). The WTI indicated the superiority of the EASS group over GUC, but
WSI weighted both these group as on par performers.
Groups such as STEM, STMM and EAIAGH had insignificant differences among
themselves and were classified as poor winter tolerant as well as poor winter sprouting
categories. The TCC was the poorest performer for both the indexes and was classified
as a winter sensitive category. Their WTI values were low due to poor WIRC yield, lesser
elongation of plant during winter month and poor WSI.
Sustainability 2022, 14, 11757 9 of 15

Table 3. Classification of sugarcane genotypic groups on the basis of WTI and WSI.

Genetic WTI Sprouts Winter

Interpretation WSI Scores Interpretation % Sprouted
Group Scores Clump−1 Elongation
Poor
STEM 0.94 Poor (0.50–1.49) 1.62 83.96 1.87 6.6
(1.01–1.99)
Poor
STMM 0.9 Poor (0.50–1.49) 1.63 76.41 2.07 5.4
(1.01–1.99)
Low temp Low temp
TCC 0.06 0.54 60.04 0.87 4.0
sensitive (<1.0) sensitive (<1.0)
Excellent winter
SSIH 5.66 4.09 Excellent (>3.0) 91.4 4.42 13.2
tolerance (>3.0)
Poor
EAIAGH 0.69 Poor (0.50–1.49) 1.54 69.94 2.13 6.6
(1.01–1.99)
Excellent winter
GUC 3.0 3.96 Excellent (>3.0) 94.96 4.16 9.4
tolerance (>3.0)
Excellent winter
EASS 3.71 5.05 Excellent (>3.0) 93.41 5.4 12.7
tolerance (>3.0)
GM 2.14 2.63 81.44 2.99 8.27
CD 0.39 0.30 2.48 0.28 0.67
CV 10.79 6.33 1.69 5.29 4.48

3.7. Identification of Winter Tolerant Clones

As per the WTI scores, seven clones, viz., AS04-635 (12.51), AS04-1687 (5.27), IK76-48
(4.39), Gu07-2276 (4.13), IND00-1040 (3.73), IND00-1038 (3.49) and IND00-1039 (3.24) were
identified excellent for their winter tolerance potential, while five clones, viz., Gu07-3849
(2.96), AS04-245 (2.92) and Co 0238 (2.54), AS04-2097 (2.13) and Gu07-3774 (2.13) were
identified good for their winter tolerance potential (Table 4). Two subtropical cultivars, viz.,
Co 98014 and Co 1148 were categorized as average winter tolerant ones.

Table 4. Classification of sugarcane genotypes into different winter tolerance categories based on
WTI scores.

Category of WTI No of Genotypes Genotype Names

AS 04-635 (12.51), AS 04-1687 (5.27), IK 76-48
Excellent winter
7 (4.39), Gu 07-2276 (4.13), IND00-1040 (3.73),
tolerance (>3.0)
IND00-1038 (3.49), IND00-1039 (3.24)
Good winter tolerance Gu 07-3849 (2.96), AS 04-245 (2.92), Co 0238
5
(2.0–2.99) (2.54), AS04-2097 (2.13), Gu 07-3774 (2.13)
Average winter tolerance
2 Co 98014 (1.84), Co 1148 (1.58)
(1.50–1.99)
Co 05011 (1.38), CYMA09-1268 (1.34),
Co 05009 (0.95), Co 06034 (0.89), Co 7717
Poor (0.50–1.49) 10 (0.86), CYM07-986 (0.82), Co 89029 (0.68),
CYMA09-1447 (0.62), CoS 767 (0.53),
Co 89003 (0.53)
CoJ 64 (0.43), CYMA10-948 (0.39), Co 0118
Low temperature (0.37), CYMA10-1460 (0.28), Co 0124 (0.17),
10
sensitive (<0.50) CoS 8436 (0.16), Co 0237 (0.15), Co 6811
(0.08), Co 453 (0.06), Co 419 (0.03)

All the excellent and good performer clones except Co 0238 are from wild or semi wild
genotypic groups, viz., SSIH (AS04-635, AS04-1687, AS04-245, AS04-2097), EASS (IK76-48,
IND00-1040, IND00-1038, IND00-1039) and GUC (Gu07-2276, Gu07-3849, Gu07-3774). In
addition to these, commercial varieties Co 98014 and Co 1148 scored average WTI values.
Sustainability 2022, 14, 11757 10 of 15

4. Discussion
The significance of MSS for PC and WIRC; WIRC and SIRC for most of the yield and
quality traits, viz., CCS (t/ha), cane yield (t/ha), NMC (0 000/ha), SCW (kg), cane diameter
(cm), brix%, pol%, purity% and CCS% indicated that the season of ratooning caused
differential phenotypic expression due to temperature and year effects. While comparing
the PC and ratoon crops, the CCS yield was significantly lower in WIRC (6.93 t ha−1 )
compared to SIRC (7.83 t ha−1 ). A similar trend was noted for cane yield, NMC, single cane
weight and cane length, indicating the ill effect of LT in the winter-initiated ratoon crop.
The varying response of sugarcane genotypes for sprouting, NMC and CCS in the ratoon
crop was also reported by earlier researchers [26,27]. In contrast to yield and contributing
traits, the WIRC recorded significantly higher pol% (14.3%) than the plant crop (14.1%).
This finding is in conformity with that of Yadava [28] who reported better juice quality
vis-à-vis sugar recovery in the ratoon crop over the plant crop.
The season of ratooning had a clear impact on the expression of important cane yield
and juice quality traits. Sugar yield, cane yield and NMC recorded significant improvement
in SIRC over WIRC, but pol% was higher in WIRC. The higher pol% in juice in the WIRC
than the SIRC is due to an increase in crop age under WIRC. Ram et al. [16], while studying
the pattern of sugar accumulation and yield performance in autumn and spring planted
sugarcane varieties, also observed that the extended period yielded higher sugar content in
the autumn crop over the spring planted crop. The LT prevailing during the sprouting time
of WIRC adversely affected the NMC (an important yield contributing trait). As a result,
the value of dependent traits, viz., cane and CCS yield, were reduced. Hasan et al. [29] also
observed that SIRC was found to be superior over WIRC in the expression of most traits
and genetic potential of genotypes.

4.1. Comparison between Different Groups for Cane Yield and Juice Quality
The commercial type groups STEM and STMM produced significantly higher CCS
yield (t ha−1 ), pol% and SCW in PC over RC. Similarly, these genetic groups (except STEM
in WIRC) produced significantly higher cane yield across the crop environments (PC, WIRC
and SIRC). The better performance of these groups for sugar and cane yield is due to their
selection history because in the past they were selected for these traits at the studied location.
Phenotypic selection in the target environment might have captured the favorable alleles in
these group of genotypes for important economic traits [30]. However, tropical commercial
cultivars (TCC) did not perform well under a subtropical environment as indicated by their
lower mean values for all the traits under all crop types studied. The poor performance
of tropical commercial varieties is due to the fact that they were selected under a tropical
environment, where LT stress does not prevail [31]. EAIAGH was another important group
that performed better over general mean for the important traits. It is to be noted that most
of the clones belonging to the EAIAGH group did not perform well in WIRC and SIRC for
CCS and cane yield. In our earlier study dealing with the E. arundinaceus, S. spontaneum
cyto-nuclear genome introgressed hybrids, a clear-cut difference in the performance for
cane yield and juice quality traits was observed among different groups and backcross
generations [31,32]. The differential response of the genotypes for ratooning ability was
also reported earlier by Bhatnagar et al. [26] and Rafiq et al. [33]; hence ratooning ability of
sugarcane cultivar is a function of genotype and environment interaction. Therefore, for
incorporating the LT tolerance such a kind of advanced generation segregating material
should be evaluated under endemic LT conditions of a subtropical climate.
The NMC population in STEM, STMM, TCC and EAIAGH was lower over the gen-
eral mean (GM) and SSIH, GUC and EASS groups, irrespective of crop environments
(PC, WIRC and SIRC). This is due to the fact that the commercial type genotypes due to
their skewed selection towards increased cane thickness and the negative linkage between
cane diameter and NMC (−0.84 correlation in our study) have reduced the NMC popu-
lation [34,35] over their wild and semi-wild companion groups such as EASS, SSIH and
GUC, which produced extremely higher NMC. In a move towards broadening the genetic
Sustainability 2022, 14, 11757 11 of 15

base of Indian working germplasm, many wild genetic resources of Saccharum spp., such as
S. officinarum, S. robustum, S. spontaneum as, E. arundinaceus and Erianthusbengalense, were
utilized [30,31,36,37]. These and other similar earlier efforts have continually enriched the
genetic diversity in the sugarcane cultivars. The wild genetic material and introgressed
hybrids had below average performance for juice quality and cane yield traits, indicating
that they need to be further crossed/backcrossed with commercial hybrids. Further, the
better performance of GUC, EASS for NMC indicates that the wild germplasm and intro-
gressed hybrids had a higher rate of genetic gain for the NMC over cane thickness and
SCW [17,38,39].

4.2. Identification of Traits for Winter Tolerance

Yield per se is the ultimate selection criteria of any crop improvement program, but
yield itself is a dependent trait controlled by several interdependent traits and is highly
influenced by environmental conditions. To identify low-temperature-tolerant sugarcane
genotypes under endemic subtropical conditions, an index called the winter sprouting
index is available [17]. However, this index gives emphasis only to sprouting potential
of the genotypes, whereas cane length, cane diameter and cane weight, etc., are the other
contributing traits to cane yield. Therefore, in the present study, while computing WTI, cane
yield, winter sprouts and winter growth are integrated together, WTI had strong positive
association with WSI (0.62 **), cane yield (0.49 **), NMC (0.57 **) and cane length (0.46 **),
whereas its association with CCS yield was insignificant (Table 2). Contrary to this SCW
(−0.35 *), cane diameter (−0.45 **) and pol% (−0.57 **) had negative association with WTI,
indicating that the WSI, cane yield, NMC and cane length were the positively influencing
traits for winter tolerant potential in sugarcane. The strong and positive association of WTI
with WSI, cane yield, NMC and cane length indicates that WTI can be an effective index in
identifying better cane-yielding winter tolerant genotypes. It is pertinent to explain that the
WSI had positive association with NMC (0.8 **) but did not exhibit significant association
with the CCS yield and cane yield. Non-significant (in PC) and weak association (in RC) of
WSI with CCS yield and cane yield was observed by Ram et al. [17]. The weak association
indicates that better winter sprouting potential does not guaranteed better sugar and cane
yield. However, the significant association of WTI with cane yield indicates that it can serve
as a better index in the selection of high yielding winter tolerant genotypes. The correlation
among the other variables confirm the findings of earlier researchers [34,35,38,39].

4.3. Classification of Groups and Clones Based on WTI and Mechanism of WT

To delineate the genotypes into tolerance or sensitive classes, various tolerance indices
are under use, viz., Yield stability index [40], Yield index [41], Stress tolerance index [42],
Geometric mean productivity [42], Stress susceptibility index [43], Mean productivity [44],
Tolerance index [44], etc. All these indices are derived from yield only, but in reality,
numerous contributing dependents/independents traits with varying magnitudes are
responsible for the final expression of the yield [45]. To classify the sugarcane genotypes
into true tolerance and sensitive classes against salinity, earlier we developed an index, viz.,
the Salinity Tolerance Index (STI) by adding up the percent reduction in the expression of
six differentially expressed traits, viz., cane yield, NMC, SCW, cane height, cane diameter,
juice extraction and tiller population under the highest stress level [45]. During LT stress,
sugarcane almost stops visible growth, but if the underground soil temp (up to 10 cm),
is above 12–15 ◦ C, the rhizosphere shows growth in terms of producing sprouts. The
winter sprouting index (WSI) proposed by Ram et al. [17] considers the sprouted clumps
and no. of sprouts per clump only, but in reality, many genotypes continue to elongate.
Further, cane yield, the ultimate trait of economic importance, is not taken into account
in WSI, so we proposed the winter tolerance index (WTI) as mentioned in the Materials
and Methods section. While classifying various categories of WTI, we considered that an
ideal tolerant genotype should have >85 percent sprouted clumps, on average 4.5 sprouts
per clump, nearly 10 cm plant elongation and should produce above average cane yield,
Sustainability 2022, 14, 11757 12 of 15

i.e., >80 t ha−1 . In such case, the WTI, will be >3.0. So, the genotypes or group of genotypes
having >3.0 WTI were categorized as excellent WT. The genotype or group of genotypes
that have >80% sprouted clumps, an average of 4 sprouts per clump, 8 cm plant elongation
and produce cane yield nearly 80 t ha−1 , has a WTI ≥2.0; we classified these into the
good winter tolerance category. In a similar fashion, the rest of the categories of WTI
were derived.
The WTI scores of SSIH, EASS and GUC groups indicate the excellent winter tolerance
potential of these groups (Table 3). Groups such as STEM, STMM and EAIAGH had
insignificant differences among themselves and were classified as poor winter tolerant.
The TCC was the poorest performer and was classified as winter sensitive because of poor
WIRC yield, lesser elongation of the plant during winter and poor WSI. These clones were
not selected for a subtropical climate; hence it was not surprising to observe poor sprouting
during winter in them [17].
As per WTI, seven genotypes, viz., AS04-635 (12.51), AS04-1687 (5.27), IK76-48 (4.39),
Gu07-2276 (4.13), IND00-1040 (3.73), IND00-1038 (3.49) and IND00-1039 (3.24) were classi-
fied as excellent, while five genotypes, viz., Gu07-3849 (2.96), AS04-245 (2.92) and Co 0238
(2.54), AS04-2097 (2.13) and Gu07-3774 (2.13) were identified as good (Table 4). All the
excellent and good performer clones except Co 0238 are from wild or semi-wild genotypic
groups, viz., SSIH (AS04-635, AS04-1687, AS04-245, AS04-2097), EASS (IK76-48, IND00-
1040, IND00-1038, IND00-1039) and GUC (Gu07-2276, Gu07-3849, Gu07-3774). Because
of good winter tolerance potential, Co 0238 at present occupies nearly 2.77 m ha (high-
est area by any sugarcane variety of the world) of area in subtropical states having LT
conditions during winter [46]. The sugarcane clones with wild relatives, viz., Erianthus
or S. spontaneum as one of their immediate parents had excellent WSI (>3.0) scores [17].
Brandes [47] and Irvine [48] suggested utilization of wild species Saccharurm spontaneum
and related genera such as Erianthus bengalensis in breeding for cold tolerance. Ram and
Sahi [39] recorded 98.61% sprouting during winter in S. spontaneum clones, 84.17% in
S. barberi clones and poor sprouting (19.24%) in S. robustum clones in a subtropical region
of India. Glowacka et al. [49] reported cold tolerance in S. spontaneum × Miscanthus hybrid
(Miscane-US84-1058). Hassan et al. [29] in a study on the ratooning potential of sugar-
cane genotypes under varying harvesting times (November, December, January, February,
March) of a plant crop observed varying response among the genotypes for number of
sprouts, millable cane, striped cane yield and sugar yield.
Genotype interaction with environmental cues influence plant development and
metabolic activity in preparation for sustained low temperatures and freezing condi-
tions [50]. A significant level of phenotypic variability and molecular diversity was reported
with in-the-wild sugarcane germplasm collected from low temperature regions Lohit and
Changlang of Arunachal Pradesh [51]. Moreover, the findings of previous studies with sen-
sitive (S. officinarum var CP69-1062) and tolerant (S. spontaneum) cultivars that responded
differently to cold stress in terms of morphology, physiology and biochemistry when com-
pared to normally grown temperature conditions and to one another indicated different
genomic structure, genetic capacity, and their other cold tolerance-related characteris-
tics [52]. Differential expression regulation of genes was also recorded in tolerant genotypes
under low temperature conditions compared to room temperature conditions [53–56].
Therefore, yield or biomass is the result of genetic component, physiological and biochemi-
cal changes that favor improved performance. Identification of winter tolerant varieties
using physiological and biochemical parameters are handy and simple [47]. In most of the
cases, many clones or varieties show tolerance to low temperature based on physiological
and biochemical traits, but they are poor in biomass accumulation. Hence, we considered
mainly the biomass as significant criteria for WTI which is the reflection of genotype and
genotype–phenotype interaction including physiological and biochemical changes.
Sustainability 2022, 14, 11757 13 of 15

5. Conclusions
The wild genetic resources of sugarcane, due to their inherent capacity to withstand
climatic vagaries, are of utmost importance for future WT breeding programprograms. The
performance of the tropical commercial cultivars (TCC) group was the poorest under a
subtropical climate, while the subtropical early (STEM), mid-late (STMM) and E. arundinaces
introgressed near commercial hybrids (EAIAGH) recorded poor to medium performance,
whereas wild species clones and introgressed groups, viz., SSIH, GUC and EASS had better
to best performance under an LT regime. The proposed winter tolerance index (WTI), which
combines cane yield, winter sprouting and winter growth of plants, classified sugarcane
clones according to their winter tolerance behavior. The SSIH (with the best WTI score),
EASS and GUC groups were identified as excellent winter ratooner, whereas STEM, STMM
and EAIAGH were identified as poor winter ratoon crops, and TCC as winter sensitive.
The wild genome introgressed hybrids (AS04-635, AS04-1687, GU07-2276, GU07-3849,
AS04-245, AS04-2097, GU07-3774) and commercial cultivar (Co 0238) had excellent/good
winter tolerance potential.

6. Suggestions and Recommendations

The researchers can use the WTI index for selection of WT genotypes in sugarcane and
other crops as it combines cane yield, cane elongation during winter and WSI. As per the
index, the genotypes or groups found to have tolerance will have better cane yield potential
as well. Co 0238, the commercial variety having good WTI, is already under cultivation.
The rest of the better WT genotypes lack desired agronomic and quality attributes, but
they can serve as parents for a directed winter tolerance breeding program based on their
breeding value.

Author Contributions: R.K. (Ravinder Kumar), M.R.M. and P.D.: investigation, data visualization,
original draft preparation; R.K. (R. Karuppaiyan) and C.A.: remove plagiarism and final editing;
N.K.: supervision; P.K.: final draft preparation; B.R.: conceptualization, supervision and editing. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to restrictions.
Acknowledgments: We thank ICAR-SBI, Coimbatore for logistic support to complete this study.
Conflicts of Interest: The authors declare no conflict of interest.

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