Genotypic variation of osmotic adjustment and desiccation tolerance in contrasting sorghum inbred lines

I

Field Crops Research, 35 (1993) 51-62

Field Crops Research

Genotypic variation of osmotic adjustment and desiccation tolerance in contrasting sorghum inbred lines J. Basnayake a'*, M.M. Ludlow b, M. Cooper a, R.G. Henzell c a Department of Agriculture, The University of Queensland, Qld., 4072, Australia b Division of Tropical Crops and Pastures, CSIRO, Cunningham Laboratory, St Lucia, Qld., 4067, Australia c Hermitage Research Station, Queensland Department of Primary Industries, via Warwick, Qld., 4370, Australia

(Accepted 9 March 1993)

Abstract Variation in maximum osmotic adjustment and desiccation tolerance were determined before anthesis for 21 lines of Sorghum bicolor (L.) Moench, selected on the basis of their capacity for osmotic adjustment and for their putative drought resistance. A reproducible and controlled imposition of gradual water stress was achieved by withholding water from plants grown in a controlled environment with a constant evaporative demand. Even though the rate of imposition of stress was lower in the second of two experiments, the maximum level of osmotic adjustment expressed by the lines was fairly consistent across both. The highest osmotic adjustment was 1.71 + 0.06 MPa for TAM422 in Exp. 2, while the lowest was 0.78 + 0.09 MPa for QL27 in Exp. 1. The difference in maximum osmotic adjustment between the highest and the lowest lines was 0.75 and 0.87 MPa, respectively, for Exp. 1 and 2. There was also variation in desiccation tolerance among the 20 lines in Exp. 2; 58% to 68% for lethal relative water content and - 3.1 to - 3.9 MPa for lethal leaf water potential. Mean values of lethal relative water content and lethal leaf water potential were, respectively, 62% and - 3.4 MPa. Maximum osmotic adjustment was inversely related to desiccation tolerance; lethal relative water content and lethal leaf water potential increased linearly as maximum osmotic adjustment increased. Thus lines with high osmotic adjustment died at a higher relative water content and lower leaf water potential, than those with low osmotic adjustment. Despite their reduced tolerance of desiccation, lines with high osmotic adjustment survived l0 days longer. In both experiments, a high level of repeatability of line mean discrimination was identified for osmotic adjustment (0.75 + 0.15 and 0.965:0.09, for Exps. 1 and 2, respectively). The repeatability on a line mean basis across experiments was also high (0.84 5: 0.07), as was the genetic correlation between the line means in both experiments (0.86 :]: 0.08 ). The high levels of repeatability suggest that the screening procedure developed in this study is robust, and that it could be used to evaluate the inheritance of osmotic adjustment in breeding populations. Key words: Desiccation tolerance; Sorghum; Variety trial; Water stress

I. Introduction S o r g h u m is g r o w n in tropical and sub-tropical areas

of the world, where available soil moisture is a major constraint o n grain yield. T o produce an e c o n o m i c yield u n d e r water-limited conditions, plants must be able to

* Corresponding author.

0378-4290/93/$06.00 © 1993 Elsevier Science Publishers B.V. All rights reserved SSDI0378-4290(93) E0022-P

52 escape, avoid or tolerate water limitation (Blum and Sullivan, 1986). Osmotic adjustment (OA) is a mechanism which alleviates some of the detrimental effects of water stress by promoting both avoidance and tolerance (Blum, 1983; Ludlow and Muchow, 1990). Osmotic adjustment is a process that assists the maintenance of turgot by accumulating solute at the cellular level, and it can reduce the effect of water stress in both the vegetative and reproductive phases of crop growth (Ludlow and Muchow, 1990; Ludlow et al., 1990). The contribution of OA to higher grain yield under water-limited conditions has been reported for wheat (Morgan et al., 1986) and for sorghum (Wright et al., 1983; Ludlow et al., 1990; Santamaria et al., 1990; Tangpremsri et al., 1991 ). The usefulness of OA as a selection criterion in a plant breeding program to improve grain yield in water-limited environments has been emphasised by various workers (Blum, 1983; Morgan, 1983; Ludlow and Muchow, 1990; Ludlow et al., 1990; Tangpremsri et al., 1991). The yield improvement for grain sorghum was 34% and 24% respectively, where water stress occurred before or after anthesis (Santamaria et al., 1990). These data show the potential value of this trait for improving yield of grain sorghum in water-limited environments through breeding for high osmotic adjustment. There is considerable evidence for genotypic variation in OA among sorghum genotypes (Ackerson et al., 1980; Shackel et al., 1982; Blum and Sullivan, 1986; Santamaria et al., 1986, 1990; Tangpremsri et al., 1991). Moreover, Blum and Sullivan (1986) reported that OA in some landraces of sorghum was as high as 1.2 MPa. While genotypic variability for OA has been reported in sorghum, there is little information available on the inheritance of the trait. In contrast, Morgan ( 1983, 1991 ) reported that OA in a selected set of wheat lines is controlled by a single gene. Desiccation tolerance (DT) is a measure of the ability of tissues to withstand loss of water (Ludlow and Muchow, 1990); Ludlow et al., 1990). It is expressed as either lethal relative water content ( RWC L) or lethal leaf water potential (geL). Species with high DT usually have high maximum OA, and they survive water stress longer than those with low levels of DT and OA (Ludlow et al., 1990). Variation in DT has been reported in sorghum ( S antamaria et al., 1986), soybean (M.M. Ludlow, unpublished data) and in Stylosanthes spp. (Fisher and Ludlow, 1983), but the inheritance of

J. Basnayake et al. / Field Crops Research 35 (1993) 51~2 this trait is not known. In order to develop a population for determining the inheritance of OA and DT, a range of potential parents must be screened for the trait. As an initial step in investigating the inheritance of OA, this study aims to characterise genotypic variation in OA and DT. A selected set of 21 inbred sorghum lines previously reported to possess different levels of OA, or putative drought resistance, are characterised for differences in OA, gel and RWC L. The relationship between maximum OA and DT is also investigated.

2. Materials and methods Genetic material

The 21 inbred lines were selected on the basis of putative differences in yield under drought conditions and their response to water stress (Table 1 ). Line 40019 was not available for the first experiment, and E57 ÷ was used instead. Cultural conditions and experimental design

Two experiments were conducted in the controlledenvironment facility at the CSIRO Cunningham Laboratory, St. Lucia, Queensland. The environmental conditions common to both experiments were: 500 / x m o l m - 2 S-1 photon irradiance, 14 h photoperiod and 25°C night temperature. In the second experiment, the day temperature was reduced (27°C instead of 30°C) and relative humidity was increased (65-75% and 95-98% instead of 60-65% and 90-95% day and night, respectively) relative to that in Exp. 1, to reduce the rate of development of water stress. Plants were grown in plastic-lined, PVC cylinders (0.25 m diameter and l m height), each containing 50 kg of air-dried soil. The field capacity of the soil was 19%, and the soil pH was 6.7-7. A randomised, complete block design, with three replicate pots per line was used for both experiments. The 20 pots were rerandomised within each of the three blocks at weekly intervals throughout each experiment. An appropriate amount of N, P, K and other micronutrients was applied 3 days before sowing, and again 24 days later to ensure that nutrient supply was not limiting growth. Six seeds were sown in each pot at the start of each experiment and plants were thinned to two per pot at the four-leaf stage. The pots were kept well-watered until this stage,

J. Basnayake et al. / Field Crops Research 35 (1993) 51~2 Table 1 Characteristics of 20 inbred lines and the hybrid E57 + of grain sorghum screened for variation in maximum osmotic adjustment (OA) and desiccation tolerance Inbred lines SC29C SC31C SC33C SC36C SC402 QL12

Characteristics

Lodging-resistantline with R.G. Henzell low level of n o n (unpublished data) senescence.

Ajebsido Dabar Kulum f .) CSM63 CSM387

Sudan origin and putative Blumand Sullivan high OA. (1986) Mali origin and putative high OA.

Blumand Sullivan (1986)

SC35C14E Lodging-resistant,and Rosenow(1977) SC56C14 highlynon-senescentlines with extensive root system. B1887 R9188 Tx2813 TAM422 KS4

Lodging-resistantand non- Rosenow (1977) senescent lines. ~.Lodging-susceptible, senescent hnes.

allow the genetic potential for OA to be expressed, and because the last measurement was taken just before plants died, the value of OA was considered to be the maximum value for each line.

Reference

t Subset of ten lines used to develop the random mating Tangpremsriet ai. population QP8R in which ( 1991) OA was studied.

QL27

53

R.G. Henzell (unpublished data)

E57 ÷

Commercial lodgingresistant hybrid.

Dekalb Shand Seed Co.

Line 40019

Drought-resistant line.

PacificSeeds (Aust.) Pty Ltd.

Measurements The measurements taken were leaf water potential ( ~ ), leaf osmotic potential (/-/) and relative water content ( R W C ) . ~ was measured using a pressure chamber (Ritchie and Hincldey, 1975) on the tip of the youngest fully-expanded leaf in the early stages of the stress cycle, and on the basal part of the leaf during the later stages of the stress cycle, because the tip of the last surviving leaf was by then necrotic. Osmotic potential was measured following the procedure outlined by Ludlow et al. (1983). Two leaf discs were punched from the leaf lamina, wrapped in a polythene film and aluminium foil, and then frozen in liquid nitrogen for 4 h. Samples were allowed to thaw for 30 min prior to measurement of H. Osmotic potential was measured using a dew point hygrometer (Flower and Ludlow, 1986). Ten scans were made continuously for each sample and the last five scans, which were more stable, were averaged to obtain H for each sample. Relative water content was measured following the procedure outlined by Barrs and Weatherley (1962). Two leaf segments (2 × 3 cm) were cut from the same leaf used for ~ and H measurements. Fresh weight of each segment was recorded before floating on deionised water and illuminated at the light compensation point for net photosynthesis for 4 h to obtain the turgid weight. The leaf samples were then dried at 80°C for 24 h and the dry weight was recorded. Relative water content was calculated using the formula of Barfs and Weatherley (1962) as RWC-

when the soil water content was raised to field capacity, and the first measurements of leaf water status were made on the unstressed plants. Gradual water stress was then imposed by withholding water. Leaf water relations were taken at 21, 36, 42 and 53 days after imposition of water stress. The final measurement was made when one third of the youngest, fully-expanded leaf was necrotic, which was approximately when 90% of the leaf area on an individual plant was dead (necrotic). Because plants were stressed slowly to

( F W - DW) (TW - DW)

× 100

where FW=fresh weight, DW=dry weight and T W = turgid weight. Relative water content was used with the measured / / t o calculate the leaf osmotic potential at 100% RWC (//1oo) (Wilson et al., 1979) as

//,oo = / / f R W C -

B] lOO-B ]

where B is the apoplastic water content, and assumed

54

J.

to be 10% for each genotype. The value of 10% was chosen for sorghum based on data of Flower et al. (1990) and Girma and Krieg (1992). A constant value of B was used, because there is no information available that B varies among sorghum genotypes and the fact that B is not significantly affected by the degree of water stress or the stress history of tropical grasses (Wilson et al., 1980). Osmotic adjustment was calculated as the difference between the two values o f H~oo measured prior to the imposition of water stress and at the severe level o f water stress ( F l o w e r and Ludlow, 1986).

Analysis

of variance

The data collected from each experiment were analysed both separately and in combination. For the combined analysis, the lines were considered as fixed effects and environment, blocks and samples as random effects. Where multiple runs of the controlled-environment experiments were conducted, slight variations in the stress conditions may have occurred. It was considered appropriate to treat this as random environmental variation. The statistical model for the analysis of variance within individual controlled-environment experiments was

Yijk = m + gi + bj + (gb)ij + sijk, where Yo'k = the phenotypic observation on sample k in b l o c k j on line i; m = overall mean; g~ = genotypic effect of the ith line with the assumption that ,~g;=0;

Basnayake et al. /Field Crops Research 35 (1993) 51-62

bj = block effect of the jth block assumed to be distributed as N(0,tr2); ( g b ) u = interaction between ith line a n d j t h block referred to as experimental error assumed to be distributed as N ( 0 , t r 2) and s u , = variation due to sampling different plants within a plot assumed to be distributed as N(0,0-~). For the pooled analysis the model was

Yijkl = m + gi + c j + (gc)ij+ ( b / c ) ~ j + e o , + Sijkt, where Y~j~=phenotypic observation on sample I in block k on line i in CE; m = o v e r a l l mean; g~ = genotypic effect of the ith line with the assumption of Zg~ = 0; cj = controlled environment ( C E ) effect of jth run in the controlled environment assumed to be distributed as N(0,0"2); ( g c ) ~ i = line X CE room interaction assumed to be distributed as N(0,tr2c); ( b / c ) , j = k t h block effect in jth CE room assumed to be distributed as N ( 0 , ~ 2) ; e~j, = error due to line by block interaction, N ( 0 , ~ ) and s o , t = s a m p l i n g variation among plants within plots, N(0,cr2). The variance components for lines (Vg), controlledenvironment runs ( a 2 ) , line by controlled environment interaction (O'gc), 2 blocks (o'2), line by block interaction (o-~) and samples ( ~ ) , were estimated by equating mean squares to expected mean squares (Table 2) and solving for variance components. The two experiments conducted in different runs of the controlledenvironment rooms were treated as different environments. The effectiveness of the screening technique and repeatability of the levels of O A in different lines were also studied.

Table 2 The sources of variation, degrees of freedom and the expected mean squares for the combined analysis of 19 sorghum lines evaluated for maximum OA in two controlled-environmentexperiments Form of analysis of variances

Degrees of freedom

Combine analysis CE

n c -- 1

Block (a)/CE Genotypes (G) G×CE G×b/CE within G × B/CE

n¢(nb - I i ng - 1 (nc-1)(ng-l) n~(ng- 1)(rib-- 1) ngnbnc ( n~ -- 1 )

Expected mean square (EMS) 2 2 2 2 o's + n s o ' e + n s n g o ' b + n s n b n g o ' c

O'2 + n~o'~ +nsngo"2 O" s2 -I- n s o " 2 ¢ -I- ~'~snbo'g2c O"s2 + r~so" 2 e + n s R b o ' g2c

"~ n s ~ b n c V g

O'~+ nso'~ O'~

O'2=sampling variation among plants within a plot, O'~=block variation, O'~=variation among controlled-environment (CE) runs, Vg= variation among lines, O'~= variation due to lines by blocks interaction and O'~g= variation due to genotypes by CE runs interaction,ng, nb, n~, and ns, are the number of lines, number of blocks, number of CE runs, and number of samples.

55

J. Basnayake et al. / Field Crops Research 35 (1993) 51--62 Repeatability (h *2) o f discrimination among lines Repeatability of discrimination among lines (Fehr, 1987) was estimated on three bases for both experiments within a controlled environment and across controlled environments; line mean basis ( h ' Z ) , a plot mean basis ~,t h pl*zx) , and an individual plant basis (h'Z). For the analysis across environments, the restricted and full models of Gordon et al. (1972) were used. The restricted model was considered to apply to experiments where all test lines could be evaluated in one session of the controlled environment. The full model was considered appropriate where all test lines could not be evaluated in one controlled-environment session and multiple runs were necessary, with lines randomised over controlled-environment sessions. These were estimated for individual experiments as *2 h,m = Vg/ ( Vg "4- 002/n b + 00~/ n~nb),

hpl.2 =

VJ ( Vg + 00°2 + 00~/n~),

h *:' = Vg/ ( Vg + 00e2 + 00~),

(1) (2) (3)

ured on a mean across samples within pots and replicates (lm) ( 1 ); a mean of samples within one pot (pl) (2); and on an individual plant basis (I) (3). Estimates of repeatability across both controlled-environment experiments relate to: a mean across samples within pots; replicates within environments (lm) (4); a mean across samples within pots (pl) (5); and on an individual plant basis (I) (6). The estimate of repeatability across controlled-environment sessions given by (7) was considered applicable to repeatability of individual plant measurements (I), where all plants could not be evaluated in one run of the controlled environment. Genetic correlation between two experiments The phenotypic correlation for OA (rp) was estimated for line means between two experiments using a linear regression model. The genetic correlation (rg) between experiments 1 and 2 for OA measured on a line mean basis was estimated using the formula given by Burdon (1977)

(rp) for the pooled analysis using the restricted model (Gordon et al., 1972) as *2 h ,m

=

Vg/( Vg '~ O'Zgc/nc + 002/n c n b + o'~/ncnbn.O,

Vg/( Vg 2 ..~ Ore 2 "3ff002s), h~2=Vg/(Vg-~-O'gc hp~l2 =

"~"O'g2 c d l - 0 °2 A t - 0 0 2 / n s ) ,

(4)

(5) (6)

and for the full model (Gordon et al., 1972)

hF

=

v d ( v g +00~c+00~ + 00go 2 + 00e2 + 002).

(7)

The terms in the estimate of repeatability are: g = variation among genotype effects, 00c2 = variation among controlled-environment runs, 002 =variation 2 = variation due to among experimental replicates, 00go interaction between lines and controlled-environment r u n , or e2 = interaction of genotypes and replicates, and 002 = sample variation among plants within plots. The terms rig, nc, nb, and ns are the number of lines, controlled-environment runs, replicates within each experiment and samples within each plot, respectively. Standard errors for the heritability models were estimated as described by Gordon et al. (1972). These estimates of repeatability of discrimination among lines relate to experiments, which were conducted within one controlled-environment facility, where OA was meas-

rg =

.

.

(h,m~l hlmE2) where rg = genetic correlation, rp = phenotypic correlation, and h~mm * * = square root of the line and hlmE2 mean repeatability for OA in Exps. 1 and 2, respectively. The method of Kempthorne ( 1969, p. 264) was used to estimate the genetic correlation between OA and desiccation tolerance. Phenotypic correlation and heritability on line means were estimated as described earlier in this paper.

3. Results Variation in maximum OA among lines Variation among the lines for the maximum OA was found in both experiments (Table 3, Fig. la). The lines TAM422 and QL27 had the highest and lowest levels of OA, respectively ( P < 0.05). The relative values of OA for the lines were consistent between both experiments (Table 3), with four exceptions. Osmotic adjustment was measured four times as water stress intensified during the continuous soil drying cycle (Table 4). The rate of development of OA and the rate of decline in qt during the stress cycle were relatively constant and similar for the two experiments.

56

J. Basnayake et al. I Field Crops Research 35 (1993) 51-62

Table 3 Maximum osmotic adjustmentof 20 inbred lines and the hybrid E57 + and the meantimeto reach the lethalplantwater statusfor two experiments Experiment 1 OA

Experiment2 OA

(MPa)

(MPa)

TAM422 Tx2813 LINE40019 CSM387 AJEBSIDO SC56C SC31C

1.53 1.17b a 1.51 1.47 1.33 1.45

1.71

B1887

1.48 b

SC29C SC35C KS4 KULUM E57 SC402 QLI2 SC33C DABAR R9188 SC36C CSM63 QL27

1.31 1.20

1.56 1.51 1.48 1.32 1.30 1.29 b 1.29 1.18

1.23

1.15

1.24 1.19 1.16 1.08 1.41b 1.16 0.86b ! .09 1.06 0.78

1.15 a 1.14 1.12 1.09b 1.06 1.06 b 1.01 0.95 0.84

53 53 53 52 52 52 52 52 52 50 50 50 50 48 48 48 48 48 43 43 43

1.23

1.24

-

0.29

0.17

-

Lines

(o)

5

Timeto reach lethal plant water status (days)

2 1 0

Mean LSD 5%

1.62b

4 27 3

~2

3, 0

58

q60

62 6466 LETHAL RELATIVEWATER CONTENT

68

70

6

5I

•

:

(c)

3 2 1 0 -4-.0

-3.8

-3.6 -3.4 -3.2 LETHAL LEAF WATER POTENTIAL(MPo)

-3.0

a Not includedin both experiments. b Lines which showed markedre-rankingbetweenExps. 1 and 2.

However, in the first experiment, the rate of decrease in ~ during the first 21 days of the stress cycle was higher than that for Exp. 2. As a result, the rate of development of OA during the early stages in Exp. 1 was relatively lower than that for Exp. 2. The higher rate of development of stress in the early stage of Exp. 1 was the result of an equipment failure. Poor air circulation in a section of the controlled-environment room caused higher air temperature and lower relative humidity. This resulted in rapid dehydration of four lines (Tx2813, B1887, SC33C and R9188) in one replication, and it contributed to a greater variation among lines in Exp. 1 compared to Exp. 2. The youngest, fully expanded leaves of B1887 and SC33C were almost necrotic at this stage, and therefore the osmotic potential of the two lines in the particular replication was

Fig. 1. Frequencydistributionsof (a) maximumosmoticadjustment, (b) lethal relativewater contentand (c) lethal leaf water potential of 20 sorghumlines in Exp. 2.

over estimated. In contrast, one third of the youngest, fully expanded leaves of Tx2813 and R9188 were necrotic at the same stage, and exhibited relatively higher osmotic potential than that in Exp. 2. The combined analysis of variance for OA revealed line by controlled environment interaction ( P < 0.05) between Exps. 1 and 2. This was associated with a degree of re-ranking of the lines for OA between experiments, weakening the association between OA expressed by the lines (Table 3) in both experiments. However, the consistency of the results for the other

J. Basnayake et al. / Field Crops Research 35 (1993) 51-62

57

Table 4 Rate of development of OA and decline in ~ since the previous measurement, and//,~o and OA on four occasions during the soil drying cycle for two experiments. Values are averaged across all lines. Final measurements were made at different times but > 40 days after water was withheld, when each line was 90% senescent Experiment

0

Days since water withheld

Rate of development of OA (MPa day -] )

Rate of decrease in (MPa day- 1)

Mean H~oo

OA

(MPa)

(MPa)

0 21 36 > 40

0 0.02 0.03 0.03

0 0.09 0.08 0.08

-

0.74 1.23 1.60 1.97

0 0.49 0.86 1.23

0 21 36 > 40

0 0.04 0.03 0.03

0 0.06 0.09 0.08

-0.81 - 1.68 - 1.91 - 2.05

0 0.87 1.10 1.24

10

20

30

40

50

60

i

[

i

i

i

i

--1

.=.

so_

2=0.4 ~

z I.,.I

Y =IZ.13X

I--

(a)

--2

~ " 65

(o) +

36.69

~ eo

,.=.

0

~= --3

~ 55

%

v-

_N 50 ,~2.0

bA

::E

__•=""=

~1.5

~

~

1.0

<

i¢ ~

•

--r

ul, 40 0.6

(b)

|

I

0.8

1.0

~t - - 70

o~0.5

2

R = Y =

0.0= 0

•

45

I 1.2

I

I

1.4

1.6

(b)

0.61 9.11x

+

50,89

•

I

10

20

30

40

50

60

65

100 p_ #90 o

=80

60

t,,.i

.J I.d

70

~6o 50

O J

•

•

"r

55 I

10

i

I

I

I

I

20

30

4-0

50

60

0.8

I

I

I

I

I

1.0

1.2

1.4

1.6

1.8

MAXIMUM OSMOTIC ADJUSTMENT ( M P o )

DAYS SINCE WATER WAS WITHHELD

Fig. 2. Change with time after water was withheld of (a) leaf water potential, (b) osmotic adjustment and (c) leaf relative water content of two lines with high (TAM422 • and Tx2813 • ) and two with low (QLI2 • and QL27 • ) capacity for OA.

Fig. 3. Phenotypic relationship between lethal relative water content and the maximum osmotic adjustment in (a) Exp. 1 and (b) Exp. 2.

J. Basnayake et al. / Field Crops Research 35 (1993) 51-62

58

0.6

MAXIMUM OSMOTIC ADJUSTMENT (MPo) 1.0 1.2 1.4 1.6

0.8 i

i

i

-3.0 •

i 2 R = 0.38 y = -O.e6X

i -

2.61 (o)

Table 5 Variance components ( 1 0 x MPa) and the repeatability estimates (h*2) for OA measured on 19 sorghum lines evaluated in two controlled-environment experiments

~-3.2

v

Exp. 1

~ -3.4 o

-5.6

~

-3.8

Components Vg 0-2

o

~_ - 4 . 0

%

-4.2

o

-4.4 MAXIMUM

0.8 -3.0

ADJUSTMENT

1.2

1.4

1.6

1.8

i

i

i

i

t

•

R 2= 0 , 5 6 Y = -O,71X

•

3.19-+ 1.36 2.97_+0.73

4.77_+ 1.63 0.73_+0.26

VgE

-

-

0-~ o-b2 VE

0.36+0.06 0.03_+0.15

-

2.56

(b]

0.49+0.15 0.50+0.15 0.75+0.15 -

h l ~ m2

hi.2 a

~- - 3 . 4 o

•

0.76_+0.14 -0.05_+ - 0 . 0 1 -

-

Repeatability h .2 ho~2

-3.2

-3.6

Across experiments

3.43+ 1.28 1.85_+0.39 0.55 ± 0.09 0.56+0.07 - 0 . 0 1 +0.03 0.03 -+ 0.02

(MPo)

1.0

" •

~

OSMOTIC

Exp. 2

•

~-3.8

0.82+0.09 0.84+0.09 0.96+0.09 -

0.53+0.07 0.56_+0.07 0.84+0.07 0.53 _ 0.07

a Full model. Vg = variation among genotypes, 0-2 = error, VgE= variation due to genotype by control environment interaction, 0-~ = variation due to sampling, ~ = variation due to block and VE = variation due to control environments, hl*2=repeatability on line mean basis, hp.2 = repeatability on plot mean basis, hE.2 = repeatability on individual plant basis.

-4.0

Fig. 4. Phenotypic relationship between lethal leaf water potential and maximum osmotic adjustment in (a) Exp. 1 and (b) Exp. 2. Regression line was fitted excluding the points denoted as O.

lines in both experiments revealed that the screening technique is quite robust. The development of OA for four contrasting lines (2 high and 2 low OA) as the plant water status fell during the stress cycle in the Exp. 2 is shown in Fig. 2. The difference in OA between the two groups became significant after 21 days of the drying cycle. The rate of increase in OA in the low group was lower than that of the high group thereafter. Moreover, maximum OA for the low group was reached by day 42, whereas the corresponding value occurred between days 42 and 53 for the high group (Fig. 2). Variation in desiccation tolerance

Considerable variations among the lines for gtL and which are measures of desiccation tolerance, were identified (P < 0.05 ) (Fig. 1b and 1c). The mean

R W C L,

2

1.8

R

=

0.82

Y

=

1.05

X -

0.09

©

~o 1 . 6 o_

< o

1.4

.~ 1.2 Ld O ~ 1.0

.... °..°°"

x w

•i "'O •

0.8 0.6 I

0.6

0.8

I

I

I

I

1.0

1.2

1.4

1.6

EXPERIMENT

IOA

(MPa)

Fig. 5. Phenotypic relationship for osmotic adjustment of 15 lines excluding the four lines stressed more rapidly in Exp. l (solid line ). Four lines are denoted as O. Dotted line shows the phenotypic relationship of 19 sorghum lines between two experiments. The R 2 value is 0.53 and Y=0.26 +0.78X.

59

J. Basnayake et al. / Field Crops Research 35 (1993) 51-62

1/"tL ( - 3.42 MPa) was consistent between the two experiments. However, the mean values of R W C L recorded in Exp. 1 and 2 were 51% and 62%, respectively. The difference in the R W C L between the two experiments may be due to the difference in the rate of development of water stress in the two experiments (Table 4). The two lines with high OA reached ~L a n d R W C L about 10 days after the two lines with low OA (Fig. 2a and 2c). The lines with the highest OA survived for 10 days longer than those with lowest adjustment (Table 3).

Relationship between OA and desiccation tolerance A linear relationship was identified between OA and the RWC L in both experiments (R2=0.43 and 0.61, Fig. 3). The estimated genetic correlation between OA and RWC L was 0.68 and 0.88 for Exps. 1 and 2, respectively. Thus, lines with high OA appear to die at a higher level of relative water content. A significant relationship was also found between OA and ~L in Exp. 1 and 2 (R2=0.36, 0.56, for Fig. 4a, 4b, respectively), but in this case plants with high OA died at a lower ~. However, this association was weaker in Exp. 1, because of the rapid dehydration of four lines in this Exp. (Fig. 4a). In the second experiment, the estimated genetic correlation between ~L and maximum OA was 0.71. While the higher RWC L of high OA lines potentially reduces the rate of survival during water stress, the), also died at a lower ~L, and after a period that lasted on average, 10 days longer than for low OA lines. Estimation of repeatability (h *2) for OA The genetic variance component (Vg) for line mean OA was greater than that for the other sources of variation in both experiments (Table 5 ). The relative magnitude of the genetic variance component was greater in Exp. 2 than in Exp. 1, because water stress developed more gradually in Exp. 2 than in Exp. 1. This markedly reduced the size of the experimental error, which increased the repeatability of discrimination among lines on mean, plot and individual plant bases in Exp. 2. The more uniform imposition of stress in Exp. 2 reduced the influence of the environmental component of variance on the degree of OA. The genetic component of the total phenotypic variation was 82%. The combined analysis showed that 53.4% and 8.6% of the variation in OA were due to the effect of line, and

line × controlled environment interaction, respectively. In general, repeatability on a line mean basis ( h i.2 m) was greater than that for the other estimates of repeatability. However, 0.84 h~*m 2 was expressed across the experiments conducted in two controlled-environmental conditions. The drop in repeatability across experiments measured on a plot and individual plant basis and for the full model emphasised the importance of controlling the rate of stress, if repeatable results are to be achieved where both series of genetic experiments are to be conducted over time and characterisation of a large number of individual plants requires the use of separate control environment facilities, with plants randomised among them. If the method of imposing water stress in Exp. 2 were used, the precision of physiological genetic studies on the inheritance of OA will be greatly enhanced.

Genetic correlation (rg) between two experiments for OA There was a significant (P < 0.05 ) linear association between Exp. 1 and 2 for the OA expressed on a line mean basis by the lines (Fig. 5). The genetic correlation between the two experiments was 0.86_ 0.09. A considerable proportion of the deviation from direct correspondence for line discrimination for OA between the two experiments was attributed to the lower precision achieved in Exp. 1. Removing the effects of four lines which were rapidly stressed in Exp. 1 (Table 3) increased the phenotypic correlation to 0.93 ___0.14 and the genetic correlation to 0.94 _ 0.09 (Fig. 5 ).

4. Discus~on

Genotypic variation for OA Genotypic variation for OA in grain sorghum has been reported by Shackel et al. (1982), Blum and Sullivan (1986), Santamaria et al. (1990) and Tangpremsri et al. ( 1991 ) with values ranging between 0.15 MPa and 1.20 MPa. This indicates that there is an opportunity for increasing the genetic expression of this trait in a cultivar development program for water-limited conditions. However, the level of OA reported in this study was higher than those reported previously for grain sorghum (Jones and Turner, 1978; Blum and Sullivan, 1986; Santamaria et al., 1990; Tangpremsri et al., 1991 ). This difference can be attributed to dif-

60

ferences in the screening method used, greater genotypic variation in the particular genotypes studied, and the lower /-/lOO values of unstressed control leaves. Comparing the OA in two sorghum cultivars, E57 ÷ and Texas671, Glinka and Ludlow (1992) found higher levels ofOA ( 1.07, 0.76 MPa) under low evaporation demand in controlled-environment conditions than that outdoors (0.59, 0.29 MPa), where the evaporation demand was greater. In the present study, the rapid rate of development of water stress in Exp. 1 did not allow Tx2813 to reach as high a value ( 1.17+0.1 MPa) as it did in Exp. 2 (1.68+0.06 MPa) where water stress was developed more slowly. In contrast, B1887 and SC33C also experienced the rapid rate of water stress in the same replication, but OA was higher in Exp. 1 than in Exp. 2. There is no obvious explanation for this behaviour. The range of OA identified in the present population were 0.75 and 0.87 MPa in Exp. 1 and 2, respectively. This is similar to the range ( 1.01 MPa) reported by Blum and Sullivan (1986) using different genetic material. The wide variation in OA makes it possible to choose parents on the basis of their level of OA, which could be used to study its inheritance. Genotypic variation f o r D T

A significant genotypic variation for DT in sorghum grown under temperature-controlled glasshouse conditions was reported by Santamaria et al. (1986). In the present study, RWC L ranged from 45% to 68%. This is larger than the range (34.5%-54.5%) reported in the previous work (Santamaria et al., 1986). However, the range of ~L ( _ 3.0 to -- 3.8 MPA) reported in this study is comparable with that reported by Santamaria et al. (1986). Crop species with greater desiccation tolerance can reach a very low level of RWC L (25%) and ~L ( _ 13 MPa) (Ludlow et al., 1983). By comparison, grain sorghum has a relatively low DT. Therefore, its ability to survive drought is mainly due to its capacity to avoid desiccation. Relationship between OA and desiccation tolerance

Flower and Ludlow (1986) found that leaves of pigeonpea died at a RWC of 32%, irrespective of the level of OA and the ~L. At the same time, ~L decreased with increasing OA in a particular line. Sorghum, however, behaves differently. Lethal relative water content was not independent of OA and qtL

J. Basnayake et al. / Field Crops Research 35 (1993) 51-62

among the 20 lines of sorghum. Rather, it increased with OA, and 61% and 56% of the phenotypic variation in RWC L and ~L was explained by the variation in maximum osmotic adjustment in Exp. 2. This indicates that lower desiccation tolerance may be a penalty for selecting sorghum lines with higher OA. However, higher OA still enhanced survival of sorghum leaves up to 10 days and it lowered the ~L by increasing RWC L per unit of ~L. High levels of genetic correlation were observed between OA and ~.L, and OA and RWC L. These demonstrate that the selection of sorghum lines for higher OA will result in a lower ~L and a higher RWC L. Therefore, this positive relationship between OA and lethal RWC has implications for attempts to combine low RWC L and high OA into one individual. Repeatability o f discrimination among lines

To study the inheritance of induced physiological characters, knowledge of the repeatability of discrimination among experimental lines needs to be established prior to estimation of components of genetic variability and heritability. This provides information on the magnitude of the sources of genetic and environmental variability, which contribute to the observed phenotypic variation among test lines. Such an understanding of these sources of variability can assist in definition of appropriate screening environments for the inheritance studies. The present study quantified the repeatability of discrimination among a fixed set of experimental sorghum lines for the maximum OA. Despite the equipment failure, the lines tested in these two experiments expressed a high degree of repeatability and a genetic correlation between two experiments, estimated on a line mean basis. The repeatability estimate in Exp. 2 was greater than that of Exp. 1. This was a result of more uniform stress conditions maintained throughout Exp. 2. The high line mean repeatability (hi'm2=0.84__0.07) estimated across experiments in two controlled-environmentconditions indicates that the screening procedure adopted in this study was robust. Some constraints have been identified in the present screening procedure. The number of plants that can be tested in a given time is restricted. This is associated with the limited space in the controlled-environment room and the requirement of a long stress cycle for the maximum expression of the maximum OA. For genetic studies, where a large

J. Basnayake et al. / Field Crops Research 35 (1993) 51-62

number of individual plants must be assessed, it is necessary to screen the plants over time in sequential runs of the controlled-environment facility. In conclusion, this experiment has demonstrated that there is considerable genotypic variation for OA and desiccation tolerance among these sorghum lines. The role of OA in grain sorghum is to maintain the RWC of leaves as the soil water deficit increases. Because of this effect, leaves are able to survive for a longer period and to a lower ~, despite the fact that they die at higher RWC. Though OA is an inducible character, a high degree of repeatability was achieved between experiments.

Acknowledgments J. Basnayake was supported by a scholarship from the Rural Industries Research and Development Corporation and Pacific Seeds (Aust.) Pty Ltd. The technical assistance of R.G. Kerslake and F. Lameree is gratefully acknowledged.

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