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Produced by the NASA Center for Aerospace Information (CASI)
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Early Warning and Crop { Condition Assessment

EW-U2-04243

JSC-17817

A Joint Program for

Agriculture and

Resources Inventory

Surveys Through

Aerospace

Remote Sensing

FEBRUARY 1982

INCREASE Or COLD TOLERANCE IN COTTON PLANT

(GQSSYPI.UM H,I lJM L,) BY MEP I QUAT CHLORIDE

(r.d4- 1J,)88) Itvt-bhAjE ut CCLE TOLERANCE IN N84-19959

(- 0TTJN P],ANT (._ (;SSY PT IIt', h 1 ESU T 1

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rLPI,^JAT C11L(jdIL)'- (AkjL1CU1LUTd1 Research

be vice) 10 j) HC 1,01/ME AO CSCL OlC U11CIdS

G3/43 00088

S. Y. HUANG, H. W. GAUSMAN, F. R. RITTIG,

D. E. ESCOBAR, AND R. R. RODRIGUEZ

U.J. DEPARTMENT OF AGRICULTURE

AGRICULTURAL RESEARCH SERVICE

SOIL AND WATER CONSERVATION RESEARCH

WESLACO, TEXAS 78596

Lyndon B. Johnson Space Center

Houston. Texas 77058
'For sale by the National Technical Information Service, Springfield, Virginia 22161

NASA — JSC

1. Report No 2. Government Accession No, 3. Recipient's Catalog No,

DISC-17817; EW42-04243

4. Title And Subtitle 5. Report date

Increase of Cold Tolerance in Cotton Plant (Gossypium

Hirsutum L.) by Mepiquat Chloride 6. Performing Organization Code

7. Author(s)

S, Y. Huang, H. W. Gausman, F. R. Rittig, D, E. Escobar,

8. Performing Organization Report No,

and R, R. Rodriguez

i0, Work Unit No,

9. Performing Organization Name and Address

USDA/ARS

111. Contract or Grant No. Soil and WAter Conservation Research

Weslaco, Texas 78596

13. Type of Report and Period Covered

12. Sppp^Qr6W Ager.cy Name and Address

1

1050 B ay Area Blvd. 14. Sponsoring Agency Code

Houston, Texas 77058

15. Suppoementary Notes

16, Abwam

Three mepiquat chloride (MC) concentrations - 40, 70, and 100 g a.i./ha - were used to

spray cotton (Gossypium hirsutum L., cultival 'McNair 220 1 ) plants to determine whether or

not MC would increase their cold tolerance. Seven to ten days after the spray, the

plants were exposed to three different cold treatments. No important difference in cold

damage was noticeg between the control and the MC-treateg plants when they were exposed

repeatedly to 4.5 C. No plants died when exposed to 0.5 C for 12 h; however, 90% of the

1st and 2nd leaves of the control plants were damaged. This was three times more

damage than those leaves of plants treated with 70 and 100 g a.i./ha MC concentrations;

60' of the control and 10-2016 of the MC-treated plants died when ^he plants were subjected to a cold hardening process with 15.5 C day (12 h) and 1.7 C night (12 h) for 10

days, and then, held at -2.2 C for 24 hours. The electrolyte leakage and reflectance

measurement data showed that the cell membranes of the MC-treated plants sustained much

less damage than those of the control. Freezing injury was easily assessed by reflectance

measurements at the 1,65-um wavelength

"Made available under NASA sponsorship

in the interest of early and wide disse lination of Earth Resources Survey

Pii, tram information and without liability

for any use made thereof."

17. Key Words (Suggested by Author(s)) 18, Distribution Statement

spectral reflectance

cold hardiness

cotton

mespiquat chloride

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No, of Pages 22, Price'

Unclassified Unclassified

JSC Form 1424 (Rev Nov 75)

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INCREASE OF COLD TOLERANCE IN COTTON

PLANT (GOSSYPIUM HIRSUTUM L.) BY

MEPIQUAT CHLORIDE

PRINCIPAL INVESTIGATORS

S. Y. Huang, H. W. Gausman, F. R. Rittig,

D. E. Escobar, and R. R. Rodriguez

APPROVED BY

Jk

Glennis 0. Boatwrig anager

Early Warning/Crop Condition Assessment Project

AgRISTARS Program

Houston, Texas

February 1982
1

INCREASE OF COLD TOLERANCE IN COTTON PLANT (GOSSYPIUM HIRSUTUM L.)

BY MEPIQUAT CHLORIDE'

S. Y. Huang, E H. W. Gausman, 3 F. R. Rittig,'

D. E. Escobar, 3 and R. R. Rodriguez3

ABSTRACT

1 Three mepiquat chloride (MC) concentrations--40, 70, and 100 g a.i./ha

were used to spray cotton (Gossypium hirsutum L., cultivar 'McNair 2201)

plants to determine whether or not MC wouldincrease their cold tolerance.

Seven to ten days after Lhe spray, the plants were exposed to three different

cola treatments. No important difference in cold damage was noticed between

the control and the MC-treated plants when they were exposed repeatedly to

4.5°C. No plants died when exposed to 0.5°C for 12 h; however, 90% of the

lst aw,l 2nd leaves of the control plants were damaged. This was three times

more damage than those leaves of plants treated with "0 and 100 g a.i./ha MC

concentrations; 60% of the control and 10-20% of the WC-treated plants died

when the plants were subjected to a cold hardening pvocess with 15.5°C day

(12 h) and 1.7°C night (12 h) for 10 days, and then, held at -2.2°C for

i 24 hours. The electrolyte leakage and reflectance measurement data showed

that the cell membranes of the MC-treated plants sustained much less damage 

than those of the control. Freezing injury was easily assessed by reflectance 

measurements at the 1.65-lim wavelength.

1 This study was funded and administered by Soil and Watf„r Conser ation

Research, Agricultural Research, Science and Education Administration,

U. S. Department of Agriculture, Weslaco, TX 78596, in cooperation with

the Texas Agricultural Ex p eriment Station, Texas A&M University System,

under Cooperative Agreement Number 58-7B30-0-171, and with the BASF Experiment Station at Limburgerhof, W. Germany under Memorandum of Understanding.

x Plant Physiologist, Texas A&M University, Agricultural Research and

Extension Center, Weslaco, TX 78596.

3 Suprvy. Plant Physiologist and Biological Technicians, respectively,

AR-SEA-USDA, P. 0. Box 267, Weslaco, TX 78596.

4 Chemist, BASF Experiment Station, 6703 Limburgerhof, Federal Republic of

Germany.

S We thank A. Peynado, Research Chemist, AR-SEA-USDA, for his help in using

the freezing chamber.
INTRODUCTION

Application of growth retardant chemicals have been shown to increase

the tolerance of some plants against eft?ec of physiological stress (6, 120

16). In laboratory experiments, Roberts (12) demonstrated that wheat plants

treated with chloromequat increased frost or cold tolerance; Freeman and

Carne (2) showed that cold injury in strawberries was reduced by using succinic acid 2,2-dimethylhydrazide (alar). Recently, prevention of chilling

injury in cotton plants by abscisic acid (ABA) was shown (11); however, the

lowest temperature used was 4°C.

Cold hardiness is often believed to be accompanied by a great reduction

in the gibberellin activity (14), and most growth retardants are known to

interfere with gibberellin biosynthesis or utilization (3). Field research

has proven that mepiquat chloride (1,1-dimetbylpiperidinium chloride) is very

effective in reducing vegetative growth of cotton (4). Since mepiquat

chloride (MC) is a growth retardant and has an antigibberellin activity (15),

its relation to cold resistactce of plants needs investigating.

We conducted this study to determine whether or not MC would protect

cotton plants from cold injury, and if so, to collect data to evaluate the

optimal concentration of MC to be used for further laboratory and field

investigations.

MATERIALS AND METHODS

Cotton plant seeds (cultivar 'McNair 220 1 ) were planted in peat pellets

(Jiffy-7 a/s Jiffy products LTD. Norway). 6 After germination, emerged

seedlings were transplanted into plastic pots containing a 1:200 mixture of

perlite and Hidalgo sandy clay loam (Typic Calciustoll). A 10-20-5 fertilizer

was added to the mixture at the equivalent of-67.2 kg/ha of N. All pots were

surface irrigated every 2 days with equal amounts of rain water.

From August 20 to October 30, 1980, the plants were kept in an open

greenhouse until they were subjected to a cola treatment, and then they were

returned to natural conditions. Day temperatures ranged from 28°C to 35%

and night temperatures ranged from 17 0 C to 24°C.

The plants were separated into three experimental groups, each with a

different time of MC application to plants and cold hardening exposure.

Each group consisted of control (nontreated) plants, which were sprayed with

distilled water, and plants hand sprayed with three concentrations of MC:

40, 70, and 100 g a.i./ha. The total volume was equivalent to 252 liter/ha.

Experimental conditions and times of application (ages) are summarized below:

6 Mention of a company name or trademark is for the readers' benefit and does

not constitute endorsement of a particular product by the USDA over others

that may be commercially available.

2.
Experi- Age (days) No. of

mental of plants days of

Group at MC No. of cold Day/night Daily photo

No. spray plants treatment temp., °C period

1 5 120 6 15.5/4.5 12 h

2 4 60 1 15.5/0.5 12 h

3 19 40 10 15.5/1.7 12 h

1 -2.2 (24 h dark

period)

Two hours after the chilling treatment, the plants were photographed with

conventional Kodacolor 6 II color negative film to record injury. Number of

cotyledons and leaves damaged were counted, and leaves of the same age were

randomly selected for light reflectance and electrolyte leakage measurements.

A Beckman Model DK-2A 6 spectrophotometer, equipped with a reflectance

attachment, was used to measure total diffuse reflectance on upper (adexial)

surfaces of single leaves over the 0.5 to 2.5-um waveband. Reflectance at the

1.65-pm wavelength was used for cold injury assessment (10).

Five discs (each 15 mm in diameter) from the leaves used for refl,e'Gctance

measurements were punched out with a cork borer, weighed, and floated on 20 ml

of distilled water. The amount of electrolyte that leaked out after 6 h at

room tem erature was measured with a Model 31 YSI Conductive Bridge (Scientific

Products;. 

The analysis of variance was applied to reflectance and electrolyte

leakage data. Duncan's multiple range test was used to compare mean differences statistically.

All plants were kept outdoors for three weeks after the chilling treatment

to observe the survival and regrowth of injured plant parts.

RESULTS

About 50 to 60% of ohe cotyledons were damaged when cotton plants were

exposed repeatedly to 4.5°C (Table 1)., None of the plants died, and cold

injury to the leaves was negligible as shown by gross examination and electrolyte leakage data (Fig. 1), but reflectance measurements did indicate that

there was a significant difference (P<0.05) in light reflectance between the

control leaves and the MC-treated leaves at the 1.65 -Um wavelength (Fig. 2).

The effect of MC on true leaves of the cotton plants was not apparent with

this level of cold treatment. ,

The Vesults from groups 2 and 3 clearly demonstrated that MC modified

the cotton plants in a way (yet unknown), which enabled the plants to be less

susceptible to cold injury. Though no plants in group 2 died, almost 90% of

the lst and 2nd leaves of the control Plants were destroyed. This was three
S,

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exposure to 15.5 0 C day (12 h) and

1.7 0 C night (12 h) for 10 days

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leaf discs from control and MC

treated cotton plants. The chilled

plants wei,: exposed to 0.5 0 C night

temperature for 12 hours. The mean

and range of conductance measurements from 3 replications are given.

tance measurements from 3 replicates are given.

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times more damage than plants treated with MC at concentrations of 70 and 100

g a.i./ha. The reason is unknown, but the younger leaves (3rd & 4th) were

damaged less than the more mature leaves (1st & 2nd) (Table 2). The injury

inflicted by cold stress above freezing was mainly leaf wilting, burn (desiccation), and abscission. The leaves from chilled plants released approximately4 times (P<0.5) more electrolytes than those from normal (nonchilled) plants,

^ indicating that they had some cold induced injury (Fig. 3). No significant

difference in electrolyte leakages between the control and MC-treated leaves

was detected and the final outcome of the leaves (abscission and death),

could not be predicted definitely by the electrolyte leakage data.

After chilling at 1.7°C for 10 nights, little damage (other than slight

t

wilting) was observed visually in group 3 plants. Group 3 plants were two

u weeks older than group 1 and 2 plants. Cold exposure for 10 days might have

+ induced some cold hardening in group 3 plants, but when the same plants were

exposed to -2.2°C for 24 hours, 60% of the control, plants were killed and

only 10 to 20% of the MC-treated plants appeared to be dead (Table 3). The

frozen dead plants showed typical freezing injury--a darkened, limp, and watersoaked appearance after thawing. The damage ratio of control to MC-treated

R plants was 3 to 1. The reflectance and the electrolyte leakage of leaf discs

from the dead and surviving plants are shown in Figs. 4 & 5. The largest difference in reflectance between frozen MC treated and untreated plants occurred

over the 1.65-um (near infrared) wavelength. The leaf discs from the dead

control plants leaked 10 times more electrolytes than those of nonchilled plants

and two times more electrolytes than those of the surviving MC-treated plants.

These results were all statistically significant (P<0.05). Freezing injury

could be assessed easily by either the reflectance or the elei;trolyte leakage

measurements; however, the differences in the degree of freezing damage among

the three different concentrations of MC-treated plants were difficult to

distinguish with either method of measurement. Some plants assumed dead in

group 3 regrew during the three-week period after freezing treatment (Table 3).

DISCUSSION

A review of several papers indicated that there is no agreement as to

the natural resistance: of cotton plants to cold temperature, especially the

threshold of killing temperature. Christiansen and Ashworth (1) reported that

approximately 40% of control cotton seedlings were killed by chilling 2 days

at 8°C. Yet in another study, only 3% of cotton seedlings died after chilling

for 4 days at 8°C (13). Guinn (5) demonstrated that chilling at 5°C for 24 hou,:s

caused cotton seedlings to wilt and increased their cotyledonary membrane's

permeability but no seedling death was indicated. Rikin et al. (11) showed

that most of the 12-day-old cotton seedlings covered with polyethylene bags

and exposed to chilling at 4°C in the dark for six days died. In our study, no

cotton plants (12 to 26 days old when exposed to cold) died even after chilling 

at 1.7°C for 10 nights or 0.5°C for 12 hours. Different experimental. conditions

--day/night temperature, photoperiod, stage of development of plants, different

strains (cultivars) and physiological conditions of plants before chilling

exposure--probably all contribute to the survival and death of plants and may

explain the discrepancies described above. For evaluating plant resistance
to chilling injury, criteria other than death of plants can be used. In our

study, the number of cotyledons and leaves that were damaged and became

desiccated were recorded, and the results showed that MC-treated plants had

their resistance to cold injury increased about threefold. The same ratio was

`. established for the resistance to freezing injury.

According to our results, 70 to 100 g a.i./ha concentrations of MC could

give the maximal protection against cold injury, but it is very important to

study the combined influence of MC spray and low temperature on the long term

effect on cotton growth and development.

The cold protection level that we found is comparable to other investigations that used ABA (11) and antitranspirants (1). The increased resistance

to cold stress induced by ABA and anti transpirants is attributed to a reduced

water loss. The increased ABA levels are believed to induce stomatal closure,

and ABA applications can result in complete and reversible stomatal closure

(8). Whether the same mechanism is involved in MC-induced protection should

be investigated.

It is generally agreed that one of the most universal measures of plant

temperature injury has been electrolyte leakage (7) and that, after severe

chilling, the tissues leak internal electrolyte and die (9). Our results

indicated that a totally damaged tissue (frozen and thawed) did leak a large

amount of electrolytes. However, the method is not specific and sensitive

enough to predict a partially damaged tissue with no visually observable injury 

that can either revive or die.

It was demonstrated through our reflectance data that detecting freeze

damage in a large scale field operation may be feasible using a technique such

as remote sensing photography.

LITERATURE CITED

1. Christiansen, M. N. and E. N. Ashworth. 1978. Prevention of chilling

injury to seedling cotton with anti-transpirants. Crop Sci. 18:907-908.

2. Freeman, J. A. and 1. C. Carne. 1970. Use of succinia acid 2,2-d.=. ethyl 

hydrazide (alai) to reduce winter injury in strawberries. Can. J. Plant 

Sci. 50:189-190. 

3. Frost, R. G. and C. A. West. 1977. Properties of kaurene synthesis from

Marah macrocarpus. Plant Physiol. 59:22-29.

4. Gausman, H. W., F. R. Rittig, L. N. Namken, R. R. Rodriguez, and D. E.. g 

Escobar. 1978. Effects of 1,1-dimethylpiperidinium chloride on plant 

growth and leaf anatomy of two greenhouse grown cotton cultivars

(Gossypium hirsutum L.). Cotton Physiology Conference Proceedings.

1978 Beltwide Cotton Production Research Conference. p. 40.
5. Guinn, G. 1971. Chilling injury in cotton seedlings--Changes in permeability of cotyledons. Crop Sci. 11:101-102.

6. Larque-Saavedra, A. and R. L. Wain. 1976, Sh!lies on plant growthregulating substances. :?,Xx. Abscisic acid au a genetic character

related to drought tolerance. Ann. Appl. Biol. 83:290-297.

7. Lyons, J. M. 1973. Chilling injury in plants. Ann. Rev. Plant Physiol.

24:445-466.

8. Malloch, K. R. and R. Fenton. 1979. Inhibition of stomatal opening by

analogues of abscisic acid. J. Exp. Botany 30:1201-1209.

9. Patterson, B. D., T. Murata and D. Graham. 1976. Electrolyte leakage

induced by chilling Passiflora species tolerant of different climates.

Aust. J. Plant Physiol. 3:435-442.

10. Peynado, A., H. W. Gausman, D. E. Escobar, R. R. Rodriguez, and M. V.

Garza. 1979. Evidence of cell membrane injury detected by reflectance

measurements. Cryobiology 16:63-68.

11. Rikin, A., D. Arsmon, and C. Gitber. 1979. Chilling injury in cotton

(Cossypium hirsutum L.): Prevention by abscisic acid. Plant & Cell

Physiol. 20:1537-1546.

12. Roberts, D. W. A. 1971. The effect of CCC and gibberellins A3 and,:,./

on the cold 'hardiness of Kharkov--22 MC winter wheat. Can. J. But.

49:705-711.

13. St. John, J. and M. N. Christiansen. 1976. Inhibition of linolenic acid

synthesis and modification of chilling resistance in cotton seedlings.

Plant Physiol. 57:257-259.

144 Waldman, M., A. Rifkin, A. Dowrat, and A. E. Richmond. 1975. Hormonal

regulation of morphogenesis and cold resistance. II. Effect of coldacclimation and of exogenous abscisic acid on gibberellin acid and

abscisic acid activities in alfalfa seedling. J. Exp..Biol. 26:853-859.

15. Willard, J. 1. 19780 PIXTM : A new plant growth regulator,. New

developments from agricultural chemical and equipment industries.

Proceedings. 1978 Beltwide Cotton Production Research Conference.

p. 78.

16. Wright, S. T. C. and R. W. P. Hiron. 1969. Abscisic acid, the growth

inhibitor induced in detached wheat leaves by a period of wilting.

Nature (London) 224:719.

*U.S. GOVERNMENT PRINTING OFFICE: 1982-589.011/519