J Sci Food Agric 1998, 78, 466È470

Eþects of Gibberellic Acid and ChlorocholineChloride on Tuberisation and Growth of Potato

tuberosum(Solanum L)Neerja Sharma, Narinder Kaur and Anil K Gupta*

Department of Biochemistry, Punjab Agricultural University Ludhiana 141004, India

(Received 9 September 1997 ; revised version received 25 February 1998 ; accepted 16 March 1998)

Abstract : Treatment of potato plants with gibberellic acid enhanced both(GA3)shoot and stolon growth and dry weight but delayed tuber initiation anddecreased tuber yield. Conversely, treatment with chlorocholine chloride (CCC)growth retardant reduced shoot and stolon growth and dry weight but promotedtuberisation. Chlorophyll a and b contents were both increased appreciably withCCC. In the tubers, CCC increased starch content by 11% compared tountreated control, whereas decreased starch content by about 13%. A veryGA3high reducing sugar content in the stem of crop indicated activeGA3-treatedhydrolysis of sucrose coming from the leaves, leading to its reduced supply to thetubers because of further possible sucrose hydrolysis while passing through thelong stolons. However, in the CCC treated crop, the higher chlorophyll contentof the leaves with reduced stolon length will promote efficient sucrose supply tothe tubers. 1998 Society of Chemical Industry.(

J Sci Food Agric 78, 466È470 (1998)

Key words : potato ; Solanum tuberosum L; tuberisation ; growth ; gibberellic acid ;chlorocholine chloride

INTRODUCTION

Potato (Solanum tuberosum L) is one of the most widelydistributed crops in the world. It is grown in the tropi-cal, sub-tropical and temperate regions and forms animportant food crop. In countries with agriculture-based economies, potato production is an importantcontributing factor to the overall agricultural produce.

Tuberisation in potato is a complex process thatresults in the di†erentiation of an underground stolon, amodiÐed stem, into a specialised storage organ, thetuber. The process is characterised by signiÐcant ana-tomical, hormonal and biochemical changes (Ewing andStruik 1992), some of the earliest including an increasein starch deposition and in percentage of cells in mitosis(Duncan and Ewing 1984). The possibility that plantgrowth substances may be involved in tuberisation in

* To whom correspondence should be addressed.

the potato plant is suggested by the responses to appli-cation of various growth regulators. Tuber initiationcan be delayed by treating plants with gibberellic acid

and partially grown tubers may respond by(GA3),ceasing to bulk and by growing out as stolons (Lovelland Booth 1967). Chlorocholine chloride (CCC), whichtends to antagonise the e†ects of gibberellins by inhibit-ing its synthesis (Harada and Lang 1965), promotestuberisation in potato (Dyson 1965 ; Menzel 1980).Other growth inhibitors like paclobutrazol, abscisicacid and tetcyclasis have also been reported to reducestem elongation but increase tuber yield and percenttuberisation (Balamani and Poovaiah 1985 ; Menzel1980 ; Kim et al 1991 ; Simko 1993). Tuber-inducingconditions have been shown to result in a lowering ofendogenous gibberellin level (Okazawa 1960). There-fore, it seemed appropriate to compare Ðeld-grownplants, treated with and CCC, with untreatedGA3plants, in terms of their morphology, dry matter, yield

4661998 Society of Chemical Industry. J Sci Food Agric 0022È5142/98/$17.50. Printed in Great Britain(

E†ects of plant growth substances on tuberisation and growth of potato 467

TABLE 1E†ect of foliar sprays of gibberellic acid and chloro-(GA3)choline chloride (CCC) on biomass partitioning of potato at

maturity (g plant~1)a

Growth Haulm Roots and T ubersregulator stolons

Èb 207É3 ^ 25É5 6É7 ^ 1É3 285É3 ^ 22É3(23É7 ^ 2É7) (0É5 ^ 0É1)

GA3 254É1 ^ 24É1c 10É8 ^ 1É4c 244É8 ^ 23É1c(31É6 ^ 4É8) (1É1 ^ 0É3)c

CCC 156É6 ^ 18É3c 5É1 ^ 0É9c 382É3 ^ 20É1c(18É6 ^ 2É2)c (0É6 ^ 0É2)

a Data are mean ^ SD of 15È18 plants taken randomly. Values inparentheses are for the dry weight and values without parentheses arefor fresh weight.b No growth regulator was sprayed.c In comparison with control P\ 0É01 (StudentÏs t-test).

and carbohydrate distribution, in order to see how thesedi†er from untreated control plants which tuberise nor-mally.

EXPERIMENTAL

Potato seeds (Solanum tuberosum cv KufriChandramukhi) were sown in the month of October inÐelds situated 247 m above sea-level at a latitude of30¡45@ N. (0É1 g litre~1) and CCC (2 g litre~1)GA3were applied as foliar sprays to the Ðeld-grown plants(till considerable run-o† occurred) starting from 5 daysafter emergence of aerial shoots, ie around about 25days after sowing (DAS). A total of seven sprays weregiven at 5-day intervals. Untreated control plants weresprayed with distilled water. Plants were harvested 85DAS and measured for height, number of main stemnodes, number of tubers, fresh weight of tubers andlength and weight of stolons.

The chlorophyll from leaves was extracted by grind-ing the tissue with 96% ethanol in the presence of 0É4 g

g~1 fresh weight (FW) tissue (Johnston et alCaCO31984). The homogenate was centrifuged at 10 000g for

15 min, and the pellet formed was re-extracted fourtimes for residual chlorophyll until all colour wasremoved. From the combined supernatants, chlorophyllwas determined using equations of Winterman and DeMots (1965) :

Chlorophyll a \ 13.70A665 ml[ 5.76A649 ml

lg ml~1

Chlorophyll b \ 25.80A649 ml[ 7.60A665 ml

lg ml~1

The soluble sugars were extracted from 1È2 g of theplant tissue taken in triplicate as described previously(Gupta et al 1993), and the reducing sugar content wasestimated (Nelson 1944). Sucrose was estimated bydetermining bound fructose after destroying free fruc-tose with 30% NaOH by using resorcinol and HCl(McRary and Slattery 1945). The amount of sucrose wascalculated by multiplying the content of bound fructoseby a factor of 1É8. From the residue left after completelyextracting the free sugars, starch was gelatinised withwater at 100¡C for 1 h. After cooling, starch was hydro-lysed completely with excess of amyloglucosidase in0É1 M sodium acetate bu†er (pH 4É4) for 6 h at 55¡C(Davis 1984). The reducing sugars were estimated(Nelson 1944) and the amount of starch was calculatedby multiplying the reducing sugar content by a factor of0É9.

RESULTS

Foliar spray of caused an increase in the weightsGA3of haulm (stem and leaves), roots and stolons (Table 1)and in plant height, number of nodes and internodelength (Table 2). plants were about 2É5GA3-treatedtimes taller than the untreated controls (Table 2). GA3promoted stem growth through both internode elon-gation and increase in node number (Table 2). Thetubers in plants were produced on longGA3-sprayedstolons (Table 3). The tuber number was similar to thecontrol in plants ; however, fresh weight ofGA3-sprayedtubers per plant was less than that of control (Table 1).The fresh weight and dry weight of roots and stolonswere also more in plants. treatmentGA3-treated GA3

TABLE 2E†ect of foliar sprays of gibberellic acid and chlorocholine chloride (CCC) on stem length, number of(GA3)

nodes, internode length and tuber number in potato at maturitya

Growth regulator Plant height Nodes per main stem L ength of middle T ubers per plant(cm) internode of main stem

(cm)

Èb 19É5 ^ 2É9 11É7 ^ 0É6 2É6 ^ 0É9 12É8 ^ 2É3GA3 48É7 ^ 10É4c 14É7 ^ 2É3 6É2 ^ 1É5c 9É2 ^ 2É9cCCC 15É4 ^ 2É8 10É1 ^ 1É1c 1É0 ^ 0É1c 16É5 ^ 3É2c

a Data are mean ^ SD of 15È18 plants taken randomly.b No growth regulator was sprayed.c In comparison with control P\ 0É01 (StudentÏs t-test).

468 N Sharma, N Kaur, A K Gupta

TABLE 3E†ect of foliar sprays of gibberellic acid and chloro-(GA3)choline chloride (CCC) on number, length and biomass of

stolons of potato at maturitya

Growth Stolon Mean Dry weightregulator number length (mg)

per plant of stolon(cm)

Èb 11É0 ^ 1É2 6É3 ^ 1É2 126É1 ^ 23É2GA3 12É2 ^ 2É3 10É2 ^ 2É3c 290É5 ^ 46É7cCCC 16É7 ^ 3É4c 2É8 ^ 0É9c 97É8 ^ 17É4c

a Data are mean ^ SD of 10È12 plants taken randomly.b No growth regulator was sprayed.c In comparison with control P\ 0É01 (StudentÏs t-test).

caused morphological abnormalities of tubers, so thatthe tubers were elongated and curved.

Conversely, CCC reduced stem length but did nothave much e†ect on the number of nodes produced(Table 2). Haulm fresh weight and weight of roots andstolons was signiÐcantly less than control (Table 1).Tubers were either sessile or present on short stolons.CCC caused an increase in both the tuber number(Table 3) and tuber fresh weight per plant (Table 1).CCC treatment produced well-shaped, medium-sizedoval tubers.

The chlorophyll content of the leaves of GA3-sprayedplants decreased more than six-fold on a per-grammeFW basis, giving the leaves a pale-green appearance,whereas it increased only moderately in CCC-treatedplants (Table 4).

Data on the changes in reducing sugars, sucrose andstarch as measured in the stem, stolons and tubers areshown in Fig 1. caused a 14-fold increase in theGA3reducing sugar content of stem. A two-fold increase inthe sucrose content of the stem in plantsGA3-sprayedwas also observed. Though the level of reducing sugarwas higher than control in the stem of CCC-treatedcrop, this was signiÐcantly lower than that of GA3-

TABLE 4E†ect of foliar spray of gibberellic acid and chlorochol-(GA3)ine chloride (CCC) on the chlorophyll content of leaves at 85

days after sowinga

Growth regulator Chlorophyll a Chlorophyll b(mg g~1 (mg g~1

fresh weight) fresh weight)

Èb 0É51 ^ 0É01 0É36 ^ 0É01GA3 0É08 ^ 0É01c 0É05 ^ 0É01cCCC 0É57 ^ 0É03d 0É39 ^ 0É05

a Data are mean ^ SD (n \ 4).b No growth regulator was sprayed.c In comparison with control P\ 0É01 (StudentÏs t-test).d In comparison with control P\ 0É05 (StudentÏs t-test).

Fig 1. E†ect of foliar sprays of gibberellic acid and(GA3)chlorocholine chloride (CCC) on the carbohydrate composi-tion of tuber, stolon and stem: RS, reducing sugar ; SUC,sucrose, ST, starch ; (3) untreated control plants ; (1) GA3-plants, (0) CCC-sprayed plants ; vertical bars showsprayedstandard deviation from the mean data obtained from three

replicates.

stem. The carbohydrate composition of thetreatedstolons was not signiÐcantly a†ected with or CCC,GA3except that both caused a slight increase in the reducingsugar content (Fig 1). In the tubers, both and CCCGA3caused a decrease in the sucrose content, and thisdecrease was more in CCC sprayed crop. Application ofCCC increased starch content by about 11%, whereas

decreased starch content of tubers by about 13%.GA3Starch content in the stem and stolons varied from 0É2to 0É3 mg g~1 FW and from 0É4 to 0É6 mg g~1 FW,respectively (Fig 1).

DISCUSSION

Gibberellins are one of the main regulators of plantgrowth and development (Hooley 1994). Exogenous

is known to decrease tuber weight andGA3tuber : shoot ratio but increase plant height and nodenumber (Menzel 1980). Several workers (Borah andMilthorpe 1962 ; Burt 1964 ; Biran et al 1972) have sug-gested that environmental and chemical factors a†ecttuberisation through their inÑuence on haulm growth,

E†ects of plant growth substances on tuberisation and growth of potato 469

which is promoted by long days, high temperatures andexogenous with a consequent inhibition or delayGA3 ,in tuberisation. Conversely, haulm growth is reduced byshort days, low temperatures and growth retardants,while tuberisation is promoted. In accordance withthese correlations, higher tuberisation in CCC-treatedplants was accompanied by a reduction in the growth ofhaulm and stolons (Tables 1 and 3). The more thantwo-fold increase in length of plants couldGA3-treatedbe due to the high levels of exogenous used in thisGA3study. However, it is not necessary that tuber initiationdepends directly on competition with haulm for nutri-ents or other requirements. In an experiment withsingle-node cuttings, when there was negligible tuberÈshoot competition, and CCC a†ected tuberisationGA3in the same way as in the whole plants (Menzel 1980).Lovell and Booth (1967) reported that application of

to potato plants at an early stage of tuber develop-GA3ment resulted in greatly reduced transfer of assimilated14C to the stolons which preceded any gross morpho-logical changes in the shoot. The same response wasgiven by disbudded plants (Booth and Lovell 1972)indicating that the main e†ect of in tuberisationGA3was apparently on the stolons themselves. However, atearly stages of development, there is rapid haulmgrowth of crops, resulting in reducedGA3-sprayedtransport of sucrose to stolons (Lovell and Booth 1967).But, at near maturity, haulm growth is almost completeand partitioning of photosynthate will be more towardsstolons, which could explain the high content of sucrosein the stolons of crops at this stage (Fig 1).GA3-treated

Foliar spray of caused deformed tubers. Some ofGA3these were sickle-shaped. Jackson and Prat (1996)reported that promoted stolon growth and causedGA3the tubers to become more elongated and elliptical thannormal. Lovell and Booth (1967) reported that thestarch level in the tubers fell with time.GA3-treatedThis was associated with the outgrowth of the tuber tipsas stolons and a failure of the tubers to bulk normally,so that, instead of getting a roughly spherical tuber, anelongated tuber was formed. They suggested that GA3treatment caused the development of the shoot sinkwhich was accompanied by a “switching o†Ï of the tubersinks by changing the nature of growth at the stolon tipsuch that starch deposition and tuber bulking were sup-pressed, leading to the eventual outgrowth of the tuberas a stolon. InÑuence of into developing tubersGA3was accompanied by a marked reduction in both theactivity of ADPG-pyrophosphorylase and the ratioof ADPG-pyrophosphorylase/starch-phosphorylase(Mares et al 1981) resulting in reduced starch synthesis.Increase in the tuber number by foliar application ofCCC has also been reported by Rex (1992).

The present results show that the chlorophyll contentdecreased under the e†ect of while CCC promotesGA3 ,the chlorophyll a and b content (Table 4). The changesin the chlorophyll level caused by these growth regula-

tors may contribute to the di†erence in the yield oftubers (Table 1). has been reported to cause aGA3reduction in chlorophyll content in S tuberosum ssp,andigena plants (Jackson and Prat 1996) and onion(Miroshnichenko and Manankov 1991). CCC has beenreported to beneÐt photosynthesis by increasing chlo-rophyll, ribulose bisphosphate carboxylase, leaf thick-ness and grain-Ðlling period (Nemchenko et al 1981 ;Tezuka et al 1989 ; Stahli et al 1995).

The carbohydrate composition of stem, stolon andtubers indicated that the e†ect of was chieÑy onGA3the reducing sugar level of the stem and that of starch inthe tubers. inhibits both tuber initiation andGA3bulking, implying that it reduces the sink strength oftubers and, as a result, sugars accumulate in the stem.Thus the application of could have direct e†ect onGA3tuber Ðlling. High reducing sugar content of the stemmight be required to meet the increased respiratorydemands of the haulm. is reported to increase theGA3activity of acid invertase in the stem internodes of Pha-seolus vulgaris, Avena and dwarf pea (Broughton andMcComb 1971 ; Kaufman et al 1973 ; Morris andArthur 1985) and may be responsible for the increasedcontent of reducing sugars in the stem. High reducingsugar content in the stem of plants, whichGA3-sprayedappears to be due to rapid hydrolysis of sucrose sup-plied by the leaves, will result in reduced availability ofphotosynthate through the stem to the stolons. Longstolons observed in crop (Table 3) willGA3-treatedfurther hinder the transport of sucrose to the tubers,resulting in reduced carbon supply to the sink and thuslower yield.

CCC is a potent inhibitor of gibberellin biosynthesis(Harada and Lang 1965) and, therefore, the observede†ects are most likely due to a lowering of the endoge-nous level of gibberellins, a class of growth regulatorsthat are known to inhibit potato tuber formation(Vreugdenhil and Struik 1989) and starch synthesis inthe tubers (Mares et al 1981). A reduced sucrose contentand high starch content is required for optimum pro-cessing quality of potato (Sowokinos 1973). Applicationof CCC, therefore, appears to improve the processingquality of potato. A high chlorophyll content of leavesand reduced haulm growth with small stolons will resultin efficient transport of sucrose from the leaves to thetubers, resulting in more carbon availability for the sinkorgans, leading to higher yield.

ACKNOWLEDGEMENTS

One of the authors (NS) is grateful to the Council ofScientiÐc and Industrial Research, New Delhi for theaward of a Senior Research Fellowship. The authors arethankful to Dr K L Bajaj, Head, Department of Bio-chemistry, Punjab Agricultural University, Ludhiana,

470 N Sharma, N Kaur, A K Gupta

for encouragement and for providing necessary facili-ties.

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