Introduction
There are several abiotic stresses responsible for
limiting the production of sugarcane (Saccharum officinarum L.),
however water deficiency is considered the main one (Endres et al. 2019). The
regions of expansion of sugarcane in Brazil are characterized by dry winter,
with periods of up to six months of water deficit quite pronounced and more
accentuated compared to regions traditionally occupied with the crop. An economical way to work around the problems caused
by water deficit in crops is to use drought-tolerant genotypes (Meena et al. 2013).
The drought is a
multidimensional stress, which causes several morphological, physiological and
biochemical effects in sugarcane plants (Abbas et al. 2014; Santos et al.
2015; Ferreira et al. 2017). Thus,
techniques that evaluate the morpho-physiological effects caused by water
deficiency can be used to differentiate tolerant and susceptible genotypes (O’Neill
et al. 2006; Silva et al. 2008, 2014a, 2018). Morphological
variables such as height and number of tillers,
volume and dry matter of roots, leaf area and stomatal density have already
been evaluated in sugarcane to differentiate tolerant and drought susceptible
genotypes (Jifon et al. 2005; Silva et al. 2008; Pincelli and Silva 2012).
Silva et al. (2007, 2018) have already studied
non-destructive physiological variables such as estimated chlorophyll content
(SPAD), maximum quantum efficiency of photosystem II, stomatal conductance and
leaf area index. Moreover, the destructive variables as chlorophyll content (by
spectrophotometry), relative leaf water content and leaf water potential were
studied by Silva et al. (2007, 2014a,
b). All of them presented a positive correlation with the drought stress.
Estimation of genetic
divergence among sugarcane genotypes has been studied aiming the selection of
progenitors for the formation of new hybrids or new segregating populations,
from divergent genotypes with higher agronomic characteristics (Alam et al. 2017). The purpose of grouping methods is to separate an
original group of observations into subgroups, with the aim of obtaining
homogeneity within and heterogeneity among the subgroups (Sneath and Sokal
1973). One of the most used optimization methods in the plant breeding area is
that of Tocher.
In
the context of changing climatic patterns in some regions of Brazil, there is a
need of development tolerant sugarcane germplasm to drought. It will also give
insight the applicability of physiological characterization of sugarcane
cultivars under water deficiency within breeding program. Therefore, the
objective of this research was to evaluate morpho-physiological traits in ten
sugarcane genotypes grown under drought conditions. Nonetheless, genetic
divergence will be evaluated between tolerant and susceptible genotypes through
the use of multivariate analyzes, considering this analysis as a useful tool in
the selection of drought-tolerant genotypes.
Materials and Methods
Site description and
experimental design
This experiment was conducted in a greenhouse at the Department
of Crop Production of the School of Agricultural
Sciences, located in Botucatu city, São Paulo state, Brazil (22º51'01" S,
48º25'55" W, 800.1 m of altitude).
Ten sugarcane genotypes
(SP91-1049, RB845210, RB855035, SP89-1115, SP80-1816, RB92579, IAC91-5155,
IACSP94-4004, CTC2, IAC91-2195) were grown under well-watered (+W) and drought
(-W) conditions. Around 22% moisture content (100% of water holding capacity) were maintained as
well-watered conditions while under drought stress (-W) conditions the pots
were maintained with 50% of water holding capacity. The cultivars IAC91-2195
and IAC91-5155 were used as a control of susceptibility and tolerance to water
deficit, respectively. The experiment was laid
out following completely randomized design under factorial arrangement and
replicated four times. Sugarcane buds with same age were extracted from healthy
plants. Three buds of each genotype were placed in pots of 22-L. Each pot
containing 20-L of Plantmax® substrate (a sterile product made from expanded
vermiculite and organic material, containing macro and micronutrients) and 55 g
of the formulated fertilizer 8-28-16, which means 4.4, 15.4, and 8.8 g of N, P
and K, respectively. After the emergence of the seedlings, there was paring,
and only the primary tiller of one plant was kept per pot.
Sampling procedures, measurements and methods
From planting up to 74 days
after planting (DAP) all pots received water in the same amount. At 75 DAP were
started the treatments +W and -W. Pot moisture monitoring was performed three
times daily by means of the ECH2O meter (Decagon, DC, Washington, U.S.A.),
coupled to Echo Check dielectric sensors (Decagon, Washington, DC, U.S.A.)
inserted into the pots. Water was replaced in an adequate amount to maintain the
water regime levels.
The measurements were
taken at 75 days after the imposition of drought stress (DAT), when the plants
were 150 days old. Initially the non-destructive evaluations were made and then
the destructive ones. Grades from 0 to 2 were attributed for each variable,
indicating the degree of tolerance to drought, being 0 (without tolerance), 1
(intermediate tolerance) and 2 (very tolerant).
The
height of the stem was determined through a tape-measure, carrying out a
measurement from the soil up to the height of the insertion +1 leaf. The number
of green leaves was determined considering as green leaves those fully
expanded, with at least 20% of green leaf area, starting at +1 leaf. For the
calculation of the leaf area (LA), measurements of the diameter and length of
the leaf blade in the middle part of the +3 leaf were carried out, using ruler
and tape-measure, and the methodology of Hermann and Câmara (1999) was used
(Equation 1):
LA = C × L × 0.75 ×
(N+2) (1)
Where C is the leaf
length +3, L is the width, the crop correction factor is 0.75, and N is the
number of open leaves with at least 20% green area.
For the counting of stomata, the methodology of Mazumdar et al. (1969) was used. The impression
was withdrawal in four regions of the middle third of +1 leaf of each cultivar,
two on each leaf face, with the impressions parallel to the leaf center rib.
The impression with the shapes of the stomata was removed with colorless enamel
and transparent tape. To realize the read, the adhesive tape was putted on a
"Neubauer Chamber" and the counting of the stomata was performed in
an area of 0.25 mm² in an optical microscope (Eclipse E200, Nikon, Shanghai,
China), using the 10x magnification objective lens.
The SPAD (Soil Plant Analyzer Development) index was obtained using a
portable chlorophyll meter (SPAD-502 Minolta Corp., Ramsey, New Jersey, U.S.A.).
The readings were performed between 8 and 10 h, in the upper, middle and lower
thirds of +1 leaf, after which the general mean of the different leaf parts was
obtained.
The maximum
photochemical efficiency of photosystem II (Fv/Fm) was measured by a portable
fluorometer (Opti-Sciences, Inc., Hudson, NH, U.S.A.). Special clips for the +1
leaf darkening were used for 30 min and subsequently the value of the variable
was obtained, according to the methodology of Maxwell and Johnson (2000), where
Fm is the maximum
intensity of the fluorescence in which all reactions of the photosystem II
(FSII) close; F0 is the
minimum fluorescence intensity, when the FSII reaction centers are open; and Fv is the variable
fluorescence, being calculated by the difference between the maximum and
minimum fluorescence intensity of photosystem II (Fv = Fm
- F0). The readings were
performed between 7 and 9 h.
The determination of the
stomatal conductance (gs,
mol m-2 s-1) and the CO2 assimilation rate (A, μmolCO2 m-2 s-1) was
performed using the Infra-Red Gas Analyzer (Li-6400XT, LI-COR, Lincoln, NE, U.S.A.).
The readings were performed in the medium region of +1 leaf and determined
between 8 and 10 h.
The
total chlorophyll content (CC) was evaluated by removing two leaf discs of 0.69
cm², with a cork borer, from the
+1 leaf, that were placed in vials containing 2 mL of N, N-dimethylformamide
(DMF). The solution was protected from light for 24 h, being subsequently
withdrawn 1 mL of the chlorophyll extract diluted in 1 mL of deionized water,
for the spectrophotometer reading at wavelengths 480, 647 and 664 nm, according to the methodology of
Wellburn (1994).
The
relative water content (RWC) was determined by weighing two 0.69 cm² leaf discs
extracted from the same +1 leaf, and the fresh tissue mass (Wf) was determined
by means of an analytical balance. The mass of the turgid tissue (Wt) was
obtained after hydration of the discs for 24 h in deionized water, followed by
removal of excess water with tissue paper from the turgid discs. The mass of
the dry tissue (Wd) was obtained after drying the leaf discs in an air forced
circulation stove oven 60ºC, for 48 h. The methodology of Jamaux et al. (1997) was used to calculate the
RWC (Equation 2):
RWC =
[(Wf − Wd) × (Wt − Wd)-1] × 100 (2)
The leaf water
potential (Ψw) was performed at the tops of +1 leaf, using
a Scholander pressure chamber (Soil Moisture Equipment, Santa Barbara CA, USA),
between 10 and 14 h, the hottest period of the day, in which the lowest values
of leaf water potential are observed.
The shoot and roots dry matter masses (SDM and RDM) were obtained at 150
DAP. The plants were separated into aerial part and roots, both parts were
conditioned in forced air circulation oven at 70ºC until constant mass. The
mass of the dry matter was determined by means of a precision scale.
Data analysis
Data were
analyzed using two-way ANOVA and, in cases of significance, the Tukey's test was followed to separate
treatments means at P ≤ 0.05). The genetic divergence among the cultivars
was calculated using the generalized distance of Mahalanobis as a measure of
dissimilarity, and the Tocher's optimization method was used as a grouping
technique. The genetic-statistical analyses were processed through the GENES
software.
Results
Morphological traits
Sugarcane genotypes, water regime and
interaction among them had significant effect on stem height, number of green
leaves, leaf area, abaxial stomatal density, and root and shoot dry weight of
sugarcane, excepting the non-significant effect of water regime on adaxial
stomatal density and interactive effect on number of green leaves (Table 1).
Except for stomatal density in
the abaxial face that showed an increase in -W treatment (Table 2); the other
traits showed a significant reduction under drought conditions (Tables 2 and
4). The highest stem height in -W was noticed for the cultivar SP91-1049 (146.7
cm); whereas the lowest (80.2 cm) was noticed for IAC91-2195 (Table 2). The
minimum percentage reduction in stem height growth, compared to +W, was noticed
for CTC2 (19.04%), while the maximum was noticed for IAC91-2195
(52.4%).
The
maximum number of green leaves at -W was observed for IAC91-2195 (6.8) and
IACSP94-4004 (6.2), while the minimum (3.8) was observed for SP89-1115 and
SP80-1816 (Table 2). The lowest reduction, compared to +W, was noticed for
RB855035 (26.0%), while the highest was noticed for SP91-1049 (50.9%). In case
of LA, the highest values in -W treatment were noticed for SP91-1049 (0.62 m²)
and SP89-1115 (0.57 m²), while the lowest were noticed for RB855035 (0.32 m²)
and IAC91-5155 (0.39 m²) (Table 2). The lowest percentage reduction was
presented in SP89-1115 (27.8%), whereas the highest was observed for IAC91-5155
(55.6%). The stomatal density in the
adaxial face had a marked effect of cultivars, because only RB855035 and
IACSP94-4004 presented a statistical difference between +W and -W (Table 2).
The maximum stomatal
densities in the abaxial face, in -W, were observed for IAC91-2195 (198) and
SP89-1115 (197); whereas the minimum stomatal densities in the abaxial face
were observed for RB855035 (168) and SP91-1049 (169) (Table 2). The highest
percentage increase of this trait under -W was noticed for IAC91-2195 (33.7%),
while the lowest was noticed for RB855035 (12.7%). The highest SDM in -W was
produced by RB855035 (182 g), while the lowest was produced by
IAC91-2195 (66.4 g) (Table 2). The lowest percentage
reduction in SDM was observed for IAC91-5155 (47.3%), whereas the highest was
observed for IAC91-2195 (75.8%). In case of RDM under -W, cultivar IAC91-2195
was on top with maximum value of 167.1 g, while the minimum values of RDM were observed in RB845210
(98.9 g) and IAC91-5155 (99.7 g) (Table 2). The
lowest percentage reduction in this trait, compared to +W, was noticed for
IAC91-5155 (20.3%), and the highest was observed
for RB845210 (48%).
Physiological traits
The sugarcane genotypes, water regime and
interaction among then had significant effect on all the studied physiological
traits as well (Table 3). The higher level of ΨwL in -W was maintained by cultivar
SP91-1049 (-1.21 Table 1:
Statistical summary of morphological variables of ten sugarcane genotypes grown
under different water regimes
Source of variation |
|
SH (cm) |
NGL (nº) |
LA (m²) |
SDAD (mm-2) |
SDAB (mm-2) |
DM (g) |
|
|
|
|
|
F values |
|
|
|
|
DF |
|
|
|
|
|
Shoot |
Root |
|
Replications |
3 |
3.59ns |
2.14ns |
0.71ns |
0.06ns |
0.24ns |
0.88ns |
6.97ns |
Genotypes |
9 |
22.97** |
9.33** |
8.57** |
3.42** |
25.61** |
41.31** |
26.41** |
Water Regime (W) |
1 |
383.74** |
169.33* |
206.24** |
3.25ns |
905.86** |
1,489.06** |
243.36** |
C × W |
9 |
3.30** |
1.89ns |
2.47* |
2.63* |
9.32** |
7.43** |
3.33** |
CV (%) |
|
8.49 |
15.90 |
17.60 |
10.59 |
3.26 |
9.69 |
12.05 |
SH: stem height, NGL:
number of green leaves, LA: leaf area, SDAD: adaxial stomatal density, SDAB:
abaxial stomatal density, DM: shoot and root dry matter mass,
ns: not significant; *:
significant at P ≤ 0.05; **:
significant at P ≤ 0.01
Table 2:
Morphological variables of ten sugarcane cultivars submitted to adequate water
regime (+W) and to water deficit (-W) conditions
Variables |
Cultivars |
||||||||||
SP91-1049 |
RB845210 |
RB855035 |
SP89-1115 |
SP80-1816 |
RB92579 |
IAC91-5155 |
IACSP94-4004 |
CTC2 |
IAC91-2195 |
||
SH (cm) |
+W |
200.1a |
138.7a |
169.7a |
178.7a |
209.7a |
172.3a |
209.5a |
151.5a |
173.8a |
168.6a |
-W |
146.8b |
99.3b |
122.3b |
126.6b |
137.8b |
122.8b |
140.1b |
109.6b |
140.7b |
80.2b |
|
NGL (nº) |
+W |
10.4a |
8.8a |
7.5a |
5.8a |
7.2a |
9.2a |
9.5a |
9.2a |
9.5a |
9.2a |
-W |
5.1b |
5.0b |
5.5b |
3.8b |
3.8b |
5.7b |
5.7b |
6.2b |
6.1b |
6.8b |
|
LA (m²) |
+W |
1.23a |
0.99a |
0.57a |
0.79a |
0.89a |
0.99a |
0.88a |
0.98a |
0.78a |
0.82a |
-W |
0.62b |
0.51b |
0.32b |
0.57b |
0.56b |
0.45b |
0.39b |
0.50b |
0.56b |
0.48b |
|
SDAD (mm-2)
|
+W |
98a |
89a |
77b |
95a |
85a |
88a |
79a |
79b |
77a |
81a |
-W |
84a |
79a |
90a |
97a |
78a |
96a |
81a |
97a |
79a |
92a |
|
SDAB (mm-2)
|
+W |
142b |
145b |
149b |
173b |
139b |
150b |
146b |
148b |
163b |
148b |
-W |
169a |
191a |
168a |
197a |
171a |
191a |
195a |
180a |
190a |
198a |
|
SDM (g) |
+W |
410.1a |
240.3a |
362.6a |
368.3a |
331.5a |
354.9a |
225.1a |
251.2a |
335.1a |
275.2a |
-W |
145.5b |
76.1b |
182.0b |
174.5b |
168.4b |
149.7b |
118.6b |
80.5b |
138.3b |
66.4b |
|
RDM (g) |
+W |
148.2a |
190.2a |
165.5a |
180.1a |
140.2a |
151.4a |
125.2a |
180.1a |
245.2a |
250.9a |
-W |
101.9b |
98.9b |
112.3b |
105.9b |
100.9b |
104.2b |
99.7b |
114.7b |
154.7b |
167.1b |
Different letters between
water regime and within the same variable indicate significant differences at P ≤ 0.05
SH: stem height, NGL: number of green leaves, LA: leaf area, SDAD: adaxial
stomatal density, SDAB: abaxial stomatal density, SDM: shoot dry matter mass, RDM: root dry matter mass
Table 3:
Statistical summary of physiological variables of ten sugarcane genotypes grown
under different water regimes
Causes of Variation |
|
ΨwL
(MPa) |
RWC (%) |
CC (µg cm-2) |
SPAD |
Fv/Fm |
gs
(mol m-2 s-1) |
A (μmol CO2 m-2 s-1) |
DF |
|
|
|
F values |
|
|
|
|
Replications |
3 |
0.23ns |
2.42ns |
1.91ns |
1.04ns |
2.65ns |
2.03ns |
0.83ns |
Cultivars |
9 |
13.49** |
6.00** |
16.14** |
14.24** |
10.94** |
22.17** |
8.23** |
Water Regime (W) |
1 |
673.03** |
528.23** |
649.21** |
719.50** |
280.25** |
1,038.22** |
996.57** |
C × W |
9 |
7.27** |
5.91** |
6.63** |
3.57** |
5.25** |
19.72** |
6.03** |
CV (%) |
|
11.38 |
2.61 |
11.65 |
6.29 |
3.49 |
20.73 |
20.20 |
ΨwL:
leaf water potential, RWC: relative water content, CC: total chlorophyll
content, SPAD: estimation of chlorophyll content via SPAD unit, Fv/Fm: maximum photochemical
efficiency of photosystem II, gs: stomatal conductance, A: CO2
assimilation rate: ns: not significant; *: significant at P ≤ 0.05; **: significant at P ≤ 0.01
MPa); whereas the minimum
value of ΨwL
was noticed for IACSP94-4004 (-1.99 MPa) (Table 4). The lowest ΨwL reduction, compared to +W,
was observed for CTC2 (51.6%),
while the highest was observed for SP89-1115 (168%). The maximum values of RWC
in the leaf under -W were observed for RB92579 (86.6) and SP80-1816 (85.4),
while the minimum value (75.7) was observed for IAC91-2195 (Table 4). The
lowest RWC reduction, compared to +W, was noticed for RB92579 (7.08%), whereas the highest
reduction was noticed for SP91-1049 (18.08%).
In case of CC in -W,
cultivar RB92579 was on top with maximum value of 44.4 µg cm-2, while minimum number of
CC of 11.1 µg cm-2 was observed in SP80-1816 (Table 4). The lowest
percentage reduction of CC was noticed for RB92579 (27.6%), and the highest was
observed for SP80-1816 (78.7%). The
highest value of SPAD index under water restriction was noticed for RB855035
(36.81), while the lowest was noticed for IACSP94-4004 (22.0) (Table 4). The
minimum percentage reduction of SPAD index was observed in RB855035 (18.2%),
whereas the maximum was observed in IACSP94-4004
(44.3%). The maximum values of Fv/Fm under -W were observed in
IACSP94-4004 (0.78) and RB855035 (0.72); whereas the minimum values of Fv/Fm were observed in CTC2
(0.67), SP91-1049 and SP89-1115 (0.68) (Table 4). The lowest percentage
reduction, under water deficit, occurred in IACSP94-4004 (3.8%), while the highest
occurred in SP91-1049 (17.07%).
The highest gs in -W (0.03 mol m-2 s-1)
was noticed for RB855035, SP91-1049 and RB92579; while the Table 4: Physiological variables of ten
sugarcane cultivars submitted to adequate water regime (+W) and to water
deficit (-W) conditions
Variables |
Cultivars |
|||||||||||
SP91-1049 |
RB845210 |
RB855035 |
SP89-1115 |
SP80-1816 |
RB92579 |
IAC91-5155 |
IACSP94-4004 |
CTC2 |
IAC91-2195 |
|||
ΨwL
(MPa) |
+W |
-0.59a |
-0.71a |
-0.81a |
-0.67a |
-0.70a |
-0.95a |
-0.78a |
-0.98a |
-0.91a |
-0.82a |
|
-W |
-1.21b |
-1.31b |
-1.84b |
-1.80b |
-1.38b |
-1.65b |
-1.37b |
-1.99b |
-1.38b |
-1.69b |
|
|
RWC (%) |
+W |
95.1a |
94.7a |
93.9a |
94.1a |
95.1a |
93.2a |
92.2a |
91.1a |
90.1a |
90.8a |
|
-W |
77.9b |
80.6b |
82.5b |
83.0b |
85.4b |
86.6b |
81.0b |
80.0b |
80.3b |
75.7b |
|
|
CC (µg cm-2) |
+W |
55.0a |
52.8a |
65.4a |
51.0a |
52.2a |
61.4a |
44.2a |
53.1a |
56.1a |
56.2a |
|
-W |
22.3b |
21.3b |
33.3b |
18.4b |
11.1b |
44.4b |
29.5b |
26.9b |
36.9b |
30.5b |
|
|
SPAD |
+W |
42.1a |
44.8a |
45.0a |
42.2a |
39.5a |
44.2a |
40.3a |
39.5a |
42.1a |
43.1a |
|
-W |
32.08b |
29.1b |
36.81b |
29.3b |
26.4b |
30.2b |
24.2b |
22.0b |
31.06b |
30.2b |
|
|
Fv/Fm |
+W |
0.82a |
0.81a |
0.81a |
0.79a |
0.76a |
0.79a |
0.77a |
0.81a |
0.78a |
0.80a |
|
-W |
0.68b |
0.70b |
0.72b |
0.68b |
0.69b |
0.69b |
0.69b |
0.78a |
0.67b |
0.71b |
|
|
gs (mol m-2 s-1) |
+W |
0.13a |
0.20a |
0.14a |
0.08a |
0.13a |
0.11a |
0.06a |
0.10a |
0.07a |
0.18a |
|
-W |
0.03b |
0.02b |
0.03b |
0.02b |
0.02b |
0.03b |
0.01a |
0.02b |
0.02b |
0.02b |
|
|
A (μmol CO2 m-2
s-1) |
+W |
14.61a |
19.92a |
14.91a |
17.73a |
19.85a |
22.11a |
18.17a |
14.22a |
22.51a |
20.11a |
|
-W |
3.79b |
3.14b |
2.01b |
2.19b |
1.52b |
3.78b |
3.84b |
2.28b |
4.47b |
3.78b |
|
Different letters between
water regime and within the same variable indicate significant differences at P ≤ 0.05
ΨwL:
leaf water potential, RWC: relative water content, CC: total chlorophyll
content, SPAD: estimation of chlorophyll content via SPAD unit, Fv/Fm: maximum photochemical
efficiency of photosystem II, gs: stomatal conductance, A: CO2 assimilation rate
Table 5: General
analysis of the cultivars with sum of the grades of all analyzed variables and
classification in levels of tolerance under
water deficit conditions
Cultivars |
Variables |
|||||||||||||
SH |
NGL |
LA |
SDAB |
SDM |
RDM |
ΨwL |
RWC |
CC |
SPAD |
Fv/Fm |
gs |
A |
Total |
|
SP91-1049 |
1 |
0 |
1 |
0 |
1 |
1 |
1 |
0 |
0 |
2 |
0 |
0 |
2 |
9* |
RB845210 |
1 |
0 |
0 |
2 |
0 |
0 |
2 |
0 |
0 |
0 |
1 |
0 |
0 |
6* |
RB855035 |
1 |
1 |
1 |
0 |
2 |
2 |
0 |
1 |
1 |
2 |
1 |
1 |
0 |
13** |
SP89-1115 |
1 |
1 |
2 |
1 |
2 |
0 |
0 |
2 |
0 |
1 |
2 |
1 |
1 |
14*** |
SP80-1816 |
1 |
0 |
2 |
0 |
2 |
1 |
1 |
1 |
0 |
0 |
1 |
0 |
0 |
9* |
RB92579 |
1 |
1 |
0 |
2 |
2 |
1 |
0 |
2 |
2 |
2 |
2 |
2 |
1 |
18**** |
IAC91-5155 |
1 |
1 |
0 |
2 |
1 |
1 |
2 |
2 |
2 |
0 |
1 |
1 |
1 |
15*** |
IACSP94-4004 |
2 |
2 |
0 |
0 |
0 |
1 |
0 |
0 |
1 |
0 |
2 |
0 |
1 |
9* |
CTC2 |
2 |
2 |
2 |
2 |
2 |
1 |
2 |
2 |
2 |
2 |
0 |
1 |
1 |
21**** |
IAC91-2195 |
0 |
2 |
0 |
2 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
6* |
*: up to 9; **: 10-13; ***:
14-17; ****: 18-21. SH: height of
stem, NGL: number of green leaves, LA: leaf area, SDAB: abaxial stomatal
density, SDM: shoot dry matter mass, RDM: root dry matter mass, ΨwL:
leaf water potential, RWC: relative water content, CC: total chlorophyll
content, SPAD: estimation of chlorophyll content via SPAD unit, Fv/Fm: maximum
photochemical efficiency of photosystem II, gs: stomatal conductance, A: CO2
assimilation rate.
lowest gs (0.01 mol m-2 s-1) was noticed for IAC91-5155
(Table 4). The smaller percentage reduction of gs was observed in CTC2 (71.4%) and the highest
percentage reduction occurred in RB845210 (90.0%). In case of A, cultivar CTC2 was on top with maximum value of 4.47 µmol cm-2
s-1, while the minimum number of A of 1.52
µmol cm-2
s-1 was observed in SP80-1816 (Table 4). The lowest percentage
reduction, compared to +W, were noticed for SP91-1049 (74.05%), while the highest were noticed for SP80-1816 (92.3%).
Genetic
dissimilarity and grouping by Tocher optimization
After the study of the response of ten
genotypes to the 14 morphological and physiological traits, the sum of the
grades obtained in each studied trait and the classification for susceptibility
or tolerance was made according to this: sum of grades from 0 to 9,
susceptible; from 10 to 13, slightly tolerant; from 14 to 17, medium tolerance,
and from 18 to 21, very tolerant (Table 5). For the adaxial stomatal density
there was no evident response, since only two genotypes had statistical
differences, therefore, this trait was not considered in the Table
with the indicative tolerance grades. The differentiation between the genotypes
through their degree of tolerance indicated that four genotypes i.e., CTC2,
RB92579, IAC91-5155 and SP89-1115 were more drought tolerant in descending
order of tolerance.
First of all, we calculated the matrices of
variances and residual covariance of the fourteen morpho-physiological
characters of the ten sugarcane cultivars to enable the calculation of
Mahalanobis Distance (D²) and schematization of the Mahalanobis matrix, of
dimension 10, according to the methodology described by Rao (1952). Thus it was
observed that the greatest genetic distances were between 6 (RB92579) and 5
(SP80-1816) cultivars, 8 (IACSP94-4004) and 3 (RB855035), and 5 (SP80-1816) and
3 (RB855035); and the lowest between 2 Table 6: Dissimilarity matrix based on Mahalanobis distance
(D²) among 10 sugarcane genotypes under water deficit conditions
Genotypes |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
2 |
110.52 |
||||||||
3 |
160.40 |
282.00 |
|
|
|||||
4 |
86.99 |
83.36 |
147.93 |
|
|
||||
5 |
75.72 |
148.86 |
302.09 |
154.10 |
|||||
6 |
216.18 |
145.27 |
183.73 |
137.63 |
309.94 |
||||
7 |
131.77 |
52.11 |
213.82 |
102.29 |
199.81 |
82.58 |
|||
8 |
207.36 |
58.11 |
303.64 |
135.87 |
251.38 |
170.32 |
92.20 |
||
9 |
92.99 |
112.31 |
117.83 |
86.23 |
204.33 |
64.10 |
64.79 |
174.76 |
|
10 |
250.09 |
96.59 |
252.85 |
150.57 |
404.43 |
140.73 |
105.56 |
76.88 |
135.46 |
(1): SP91-1049; (2):
RB845210; (3): RB855035; (4): SP89-1115; (5): SP80-1816; (6): RB92579; (7):
IAC91-5155; (8): IACSP94-4004; (9): CTC2; (10): IAC91-2195.
Table
7: Relative contribution (RC) of 14
morphological and physiological variables for the calculation of the genetic
dissimilarity of 10 sugarcane genotypes under water deficit conditions
Variables |
S.j |
RC (%) |
Shoot dry matter mass |
1,656.60 |
23.41 |
Chlorophyll content |
1,070.40 |
15.12 |
Abaxial stomatal density |
692.63 |
9.79 |
Estimation of chlorophyll
(SPAD) |
683.05 |
9.56 |
Water potential |
576.59 |
8.15 |
Stomatal conductance |
530.25 |
7.49 |
Green leaves |
491.26 |
6.94 |
Foliar area |
424.12 |
5.99 |
Root dry matter mass |
225.56 |
3.19 |
Adaxial stomatal density |
189.42 |
2.68 |
Fv/Fm |
154.73 |
2.19 |
Relative water content |
153.99 |
2.18 |
Stem height |
131.54 |
1.86 |
CO2 assimilation rate |
96.38 |
1.36 |
S.j: Contribution of the variable X
to the value of Mahalanobis distance between cultivars ie., i',
RC: Relative Contribution, Fv/Fm:
maximum photochemical efficiency of photosystem II
Table
8: Grouping by Tocher optimization of ten
sugarcane cultivars under water deficit conditions
Groups |
Cultivars |
I |
SP91-1049, SP80-1816 |
II |
S989-1115, CTC2 |
III |
IACSP94-4004, IAC91-2195 |
IV |
IAC91-5155 |
V |
RB855035 |
VI |
RB92579 |
VII |
RB845210 |
Groups and cultivars in bold indicate drought tolerant and genetically distant cultivars
(RB845210) and 7
(IAC91-5155), 2 (RB845210) and 8 (IACSP94-4004), 6 (RB92579) and 9 (CTC2), and
7 (IAC91-5155) and 9 (CTC2) (Table 6).
The calculation of the
contribution of each evaluated trait, as well as its relative contribution to
the calculation of genetic dissimilarity, revealed that the four traits with
the greatest contribution in the calculated value of genetic dissimilarity
between the accesses were shoot dry matter (23.41%), chlorophyll content (CC)
(15.12%), abaxial stomatal density (9.79%) and SPAD index (9.56%) (Table 7).
The information obtained through the genetic dissimilarity matrix enabled the
grouping of the 10 cultivars studied in seven distinct groups (Table 8).
Discussion
Drought stress impaired
sugarcane growth and development, and divergent genotypes behaved differently
due to their different genetic makeup (Guan et
al. 2015; Chen et al. 2016). In
sugarcane, the water deficit promotes restrictions on cell division, number of
green leaves, leaf area, elongation rate of leaves and stems,
emission of new tillers, and on the accumulation of dry matter; reflecting
penalty in the final yield (Inman-Bamber and Smith 2005; Vieira et al. 2014). Likewise, significant
reductions in the growth of all studied genotypes under water stress conditions
were observed in this study (Table 2).
According
to Inman-Bamber (2004), the number of green leaves can be used as an indicator
of the effect of this stress on sugarcane. In this sense, leaf area also can be
an indicative of tolerance to water deficit, since sugarcane cultivars with
greater number of green leaves have larger leaf area. Cultivars
considered susceptible in this work had greater
reductions of leaf area in water deficit conditions,
which lead to decrease in interception of solar radiation,
transpiration, stomatal conductance and photosynthesis. In addition to early
leaf senescence, all this in turn decreases the CO2 assimilation and
thus the accumulation of biomass (Santos and Carlesso 1998; Ferreira et al. 2017; Silva et al. 2018).
In case of stomatal
density, Bertolino et al. (2019) affirm
that tolerant plants may respond to water deficiency by emitting new leaves
with greater stomatal density, but with smaller diameter of stomata. This
allows the air around it to become more humid, increasing the resistance to air
movement of the layer adjacent to the leaf epidermis, thus avoiding further
damage to gas exchange. However, for SDAD the water regime had little
interference, so it allowed inferring that this trait does not receive a
pronounced interference of the water deficit. From
this, it can be inferred that the number of green leaves, the leaf area and dry
matter mass were traits indicative of water deficiency, since cultivars
considered tolerant, such as CTC2, SP89-1115 and RB92579, performed
well in these variables. However, the stem height, even varying among
cultivars, did not follow a standard that could be related to a level of stress
tolerance, and the results showed that the use of adaxial and abaxial stomatal
density is not recommended as an indicative of tolerance to water deficit. The chlorophyll is the main pigment responsible for
the capture of the light energy used in the photosynthesis process; and
chlorophyll contents in sugarcane cultivars, though cultivars had divergent
response, were decreased under drought stress (Table
4). The decrease in chlorophyll content under water deficit is considered a characteristic
symptom of oxidative stress caused by photo oxidation and pigments degradation
(Farooq et al. 2009), more expressed
in susceptible cultivars (Chen et al.
2016). Silva et al. (2014a) and Kumar
et al. (2019) also verified values
lower than 40 for SPAD index in sugarcane under water deficiency as were
observed in this study. Silva et al.
(2007; 2011; 2018) affirmed that SPAD index readings
lower than 40 evidenced the beginning of chlorophyll degradation due to water
restriction, thus affecting the photosynthetic apparatus of sugarcane.
According to Silva et al. (2007) and Silva et al. (2018), ability of sugarcane
plants to maintain high Fv/Fm value under water
deficiency indicates the maintenance of high radiation use efficiency and
carbon assimilation. The cultivars considered tolerant which had lower
reductions of Fv/Fm; this suggests a greater
capacity of these cultivars to resist to photoinhibitory conditions under water
deficiency. Thus this trait was reliable for differentiating between
drought-tolerant sugarcane cultivars, with the benefit of being
non-destructive. Stomatal closure, strategy used by plants to reduce water loss
through transpiration, compromises photosynthetic carbon assimilation, due to
the reduction in CO2 influx. In this study, all cultivars showed
reduction in gs when
submitted to stress, although combined with a strong varietal effect and great
genotypic variability, as verified by Santos et al. (2015). This suggests that the response is intrinsic to each
cultivar. Despite, CO2 assimilation rate was efficient in
differentiating cultivars between tolerant and susceptible, and can be used in
studies as a tool indicative of tolerance.
Significant reductions in
ΨwL were also
found by Medeiros et al. (2013) in
sugarcane under water stress. According to results of this study, cultivars
which maintained higher levels of ΨwL, obtained higher stem height and dry
matter mass, as CTC2 and IAC91-5155. The reduction of RWC of the leaves is considered as a
good indicator of plants water conditions under water stress, once elementary
changes in water balance induce cell damage (Hussain et al. 2018). These changes can paralyze the growth and even lead
to death of plants (Zhang et al.
2014). In this context, plants that can maintain higher values of ΨwL and RWC under
water deficiency are considered tolerant (Santos et al. 2015; Silva et al.
2018). Thus, ΨwL and RWC could be used as indicators to
select drought tolerant sugarcane cultivars.
In the plant breeding area
there are methods of grouping or projections of distances in two-dimensional
graphs, which are used by breeders, based on the coordinates obtained from the
chosen genetic dissimilarity measure (Cruz and Carneiro 2006). Among the
optimization methods most used in plant breeding, stands out the one of Tocher,
that is used as an optimization grouping technique and has as basic principle
to maintain homogeneity within and heterogeneity between the formed groups (Rao
1952). The studied cultivars grouping
showed the first two groups, I and II, (closest to each other) with the three
"SP" cultivars and with the only "CTC" cultivar present in
the experiment. It is emphasized that the sugarcane breeding program
"SP" (COPERSUCAR) started to adopt the CTC acronym (Sugarcane
Technology Center) since 2004, so it can be considered the same program due to
the same germplasm bank and the same cultivar selection methods and objectives.
While in groups III and
IV, furthest from the first two and closest to each other, there are the three
"IAC" cultivars, and the groups V, VI and VII include the three
"RB" cultivars; evidencing the genetic distance between the groups
and, consequently, between the cultivars. Based
on the grouping obtained, group II contains two cultivars evaluated as
tolerant, SP89-1115 and CTC2, and the other two tolerant are IAC91-5155 and
RB92579 from groups IV and VI, respectively.
Conclusion
The morpho-physiological
traits of sugarcane were efficient to differentiate tolerant and susceptible
genotypes to water deficiency. Under water deficiency, the genotypes that stood
out for most of the morpho-physiological variables were RB855035, SP89-1115,
SP80-1816, RB92579 and CTC2. The multivariate analysis and genetic grouping
showed that the most promising crosses were: SP89-1115 and CTC2, both belonging
to group II, crossed with RB92579 or IAC91-5155 of groups VI and IV,
respectively. The descendants of these crosses might be able to obtain better
results under drought conditions.
Acknowledgements
To the National Council for Scientific and Technological
Development (CNPq, Brazil) through “Productivity in Research” fellowship for
MAS (Proc. 305952/2018-8).
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