Bioscience R..,SG2rch· Communications

Vol. 13, NO.5, October 3',. 2001Printed in Nigeria

BRC 2000006/13505

0795-807212'X;; 1·1200 .•.0.00~ 2001 Klobex Aeade:mc Publishers

Evaluation of Different Formulations of Weak Acid

Solution as Preservative Medium for Sugarcane Stalks

I I" 1G. Olaoye , O.B. Bello and F.A."'A?ekola-

IDcpnrtment of Crop Production, University of lIorin, P.M.B. 1515, florin

)Department of Chemistrv. University of Ilorin, P.M.B. 1515, !lorin.

(Receivl:d .;QflUary 21, i.aOO)

ABSTRACT: Two separate experiments were conducted at thc sereenhouse of the Unilorin Sugar Resenreh Institute(USRI) IIorin, to investigate the optimum concentration of a weak acid solution required fo'r the prescf\'aLion of cut

sugarcane stalks during hybridizntion. In the first experiment. stalks of two (2) sugarcane varieties (LS 1-047 and LS1­

057) which were at the flowering. phase were immersed in three (3) eonecntrations(Blank, O.004M nnd 0.006M

respectively) of sulphurous acid (H2SO]) solution. In the second experiment, stalks of three (3) varieties viz: LSI-047,

LSI-057 and C06806 at the vegetative phase and those of LSI-047, LSI-050, LSI-054, LSI-057, B6609 and'C06806 at

the Dowering phase were also immersed in four (4) concentrations (i.e. Blank, 0.0021\1, 0.004M and O.OO(,M

respectively) of sulphurie acid (H2S04) solution.

Our results showed a rapid decline in the physiologienl activities of the sugarcanc stalks regardlcss of the

concentration and/or the source of the weak acid used as preservative solution. Flowering sequence was also disrupted

in the flowering stalks since none of the varieties could proceed to tile next ,phase, indicating that the stock solutionswere toxic to the test varieties. However, rooting and side shoot emergence (beginning from the riftll and ,cvcnth day

rcspeetively) were observcd ill the sugareanc stalks at both the vegetative and Oowerillg ph,lsl's or diiTercnleoneelllralion of the weak 1-1250 •. indicating Ih,1I the: i'nrlJ'ulal ions 'frolll 112SO., were less tOXIC {O ti,l'. l' '" 1-::. d"'11 {I")';l'or the 1-1)50. where the test varie{les nl:ilhl:r r()o(ed nor produce sid\' ,hm)l.

i\ILllllllgh 1110 (2) or the: \'arie:lIl'S (L,SI-O'17 ,md 1..'()ll~()I,) ,'ppe"rl'" III PUSs,'ss greater {,lkral":C: k.\cl I·.' Ii;.: .!,!!"<',.:l'!

forllllliatiolls of the H)50., the locals (LSI) ilppC'lIl'.lI 10 POSS'5S g,cillcr \llk'rilllel' capaeit~, 10 tilt, Silld, ,·;,.,I;'!I,>I, 1:';11' lil<.'

exoLic judging by their overall performallce.

Introduction

The most convenient hybridization method ill sugarcane (Sac;cahrum ojjicnarum L.) involves the use of

weak sulphurous acid (H2SO}) solution which serves as preservative medium for cut flowering stalks

during crossing and fuzz (seeds) rupening. When used in conjuction with the breeding laniern (specially

constructed rectangular enclosures made up of cloth materials and supported by metal wires), the technique

501

... ~ .._~---...__ .. -......•.. _-" ..••..-

permist the sugarcane breeder to set up planned crosses between male sterile clones (as females) and malefertile clones (as males) without fear of contamination from wind-borne pollen.

In most sugarcane breeding stations where the H2S03 solution is used as a preservative medium (lorexample, Hawaii and Florida, U.SA.), the active ingredient which is the liquefied sulphur dioxide (S02) is

manufactured industrially and supplied to the breeding stations· upon request (Miller, personalcommunication). Since this facility is presently unavailable in ~igeria, our present hybridizationprogramme has been limited mostly to collection of fuzz from (i) open pollinated- anows (flowers) offemale clones of planned crosses or (ii) the recently proposed modified polycross method of sandwichingfemale clones between identified male clones whose flowering period could be synchronized (Olaoye,] 996). The necessity to maintain sufficient isolation distance from the nearest sugarcane field (so as to

ensure the absence of contamination from unwanted pollen) limits the number of such crosses that can beestablished at a time. Our experience with marcotting method has also shown that it is only feasible withfreely flowering varieties. This is because with sparsely flowcring clones, many of the marcatteu stalksmay not flower thus limiting the number of crosses that could' be made. Furthermore, it has beenestablished that the ratio of the female to malt: clones in our germplasm is very low (Olaoye, unpublisheddata). It therefore follows that in order to fully utilize the genetic potentials of our germ plasm accessions, itis necessary to explore the possibility of locally producing the required preservative solution The

advantage of such formulation include opportunity to (i) ulilize thl: superior clones in series of crosses IIImaximize genetic variability for characters of interest and also crcate new ones; (ii) gain inllmll:lliol1 onthe mode of inheritance of certain qualitative and quantitative characters, including resistance to prevalent

disease and insect pests, in order to determine lhe type of breeding system to employ in improving the

overall productivity of the crop and (iii) set up cro~ses aimed at either correcting certain genetic defects or

upgrade other characteristics presently lacking in the existing commercial varieties.Apart from the success with H2SO] reported in the literature (Warner, 1953), there hus been no other

report of any formulation for maintaining sugarcane stalks in a physiologically active state for the purposes

of hybridization. This study was therefore designed to investigate the effect of different formulations ofweak acid solution (including H2SO.1) on the survival and physiological activities of sugarcane stalks aimedat developing an appropriate medium for maintaining flowering stalks in a viable state during hybridizationprogramme.

Materials and Methods

"'rhoratory preparatio/l (~ld{fferentjiJf'll1u/atiolls of the weak acid sO/lItioIlS: Analytical procedure ji),. theproduction of H}S(),:

About fifty grams (SOg) of zinc (Zn) metal was placed in a conical ·flask connected via the ueliverytubes to two other conical flasks connected in series into which 200ml of distilled water were added.

Approximately, IaOrnl of dilute tetra-oxo-sulphate VI acid (20% or'the H2S04) was introduced into thereactor which contains excess amount of zinc metal. This generated a stream of sulphur IV oxide (S02) gas

which was then absorbed into the other two conical flasks containing distilled water until the reaction wascompleted. The process was repeated several times to generate sufficient quantity of the solution requiredfor the study.

Determillatiofl of the molarity of H 2S0; alld the ti{ratiofl procedure:

Ten milliliters (10ml) of the H2SO] was added to the conical flask using 10mi pipette. Theconcentration of the stock solution (i.e. HJS03) was determined by simple titration against 0.05M Na2C03

and using phenophtalein as indicator.

Determination of the molarity of H ."!SO, solutioll:

After the molarity of the dil. H2S04 solution has been determined as described forH2SO], IOml ofH2S04 was added into the conical flask using lOrnl pipette. A drop of phenophtalein as indicator was

502

dispensed, then O.05M of Na2CO} was gradually introduced into the j~ask until the solution turned pink

Thereafter, the volume of Na2CO} at the point of change was determined ancLrecrn:ded. A range ofconcentrations of H2S04 (O.002M, O.004M and O_006M respectively) were thus prepared for the experiment

by dilution using doubly distilled water.

Experimcma/ procedure:

Two separate-experiments were conducted at the screenhouse of the Unilorin Sugar Research Illstitute

(USR1). The experimental materials included stalks of sugarcane varieties at two distinct growth phases(i ..e. vegetative and flowering respectively) The varieties used w~re part of the flowering types among the

48 sugarcane germplasm accessions being maintained at the USRI research farm. For each experiment,stalks of the clones were prepared in the morning by cutting them··above the ground level and the cut end of

each stalk were immediately wrapped with polythene sheet. This; was done to prevent air from enleringinto the capillary vessels. The stalks were then transported to the :screenhouse where the experimem wasset up. Preparatory to the set up of the experiment, dried leaves and leaf sheaths were removed from thestalks. Similarly, dry tips of each leaf was cut with the aid of a small scissors. The polythene sheets werethen removed and a smooth cut end made (where necessary) folloWCi;dby inserting the cut end infO jars.containing the stock solutions. For each experiment, doubly distilled water (i.e. blank) served as thecontrol.

Experiment I:

The materials used comprised of flowering stalks of two (2) varieties viz: LSI-047 and LSI-054respectively which were at the initiation phase of flowering. The two ,clones were selected for the study

since at the time of setting up the experiment, they were the only male and female clQnes with sutticient.flowering stalks. The cut end of each stalk was inserted into jars containing SOOml each of the

.. concentrations of H2SO] (blank, 0.004 and O.006M respectively) and each treatment was replicated twice.The stalks were then held in erect position with the aid of stick bars. The level of the solution was

monitored regularly and made constant by replenishing the stock with the equal amount that was necessary.

Data co/lee/iol/:

The general physiological conditions of the sugarcane stalks in each solution was monirored daily andthe following indices were used to assess the performance of the clones

(i) the 'stay green' assessmenl (i.e. visual appearance);(ii) the rate at which leaves withered (an assessment of loss of physiological. activity in the

. a/reeted leaves) and(iii) progressive development of the flowering phases.

Experiment II

The general outline of the materials and methods are essentially similar to that of experiment { exceptthat 0) a different source of dilute acid (H2S04) was used and (ii) additional v~ieties at both the

vegetative and flowering phases were used.For the vegetative phase, sugarcane stalks of similar age (based on the number of internodes/stalk) were

selected from vars. LST-047, LSI-050 and C06806 and prepared as described for experiment r. Withrespect to the flower.ing phase, additional varieties (vi;,:: LSJ-047-LSJ~054 and B6609) were included tomake a total of five varieties for this phase. The materials were assessed in fourdifferent concentrations ofthe dil. H2S04 (biank, 0.002M; O.004M and 0.006M respectively) and set up as described in experiment 1The level of each stock solution was also maintained similarly as for experiment [ and the same set of datacollected on all the varieties.

503

Results. .

Generally, there wa~ a gradual decreasc in thc le\lcl of the stock solutions (except in lhe COllillll)

beginning from two days after the commencement of the experiments and this 'is an indiclltion that probahiythe preservative medium has activation and catalytic effects on the stored nutrients and other metabolites inthe cane staJks.

fxperimef// I

Slay green assessment (visual appearallce):

Beginning /Tom two days after the commencement of this study, decline in physiological activities were

observed in both varieties as their leaves began to wither. The withering began from the older leaves whichprogressively turned form the normal green colour to brown and drying /Tom the tip of each leaf

Leaf reten/ioll capaCity:

The result of the leaf retention capacity of the two varieties (Fig:" 1) reveaJed a rapid decline in thenumber of leaves as they withered and died prematurely especially in Jne' control and 'in O.006M H2SO.1

respectively. However, var. LSI-047 exhibited a greater tolerance capacity than var, LSl-054 especially atthe lower concentration (0.004M) ofH1SO" as the two inner leaves survived up"tilJthe J5th day in thismedium.

Progressive developmef/t (~fflowerif/X phase:

In sugarcane, four distinct phases of flowering IS recognizable and the sequence is as follows: .

initiation~ flaggin'g -Hipping ---~arrow emergence. Although the two varieties were at the initiation

when the experiment was set up, neither of the two could proceed to the next phase even at the lowerconcentration of O.004M (H2SOJ) and this may be due to disruption in the hormonal system of the plantsresulting from toxicity ofthe solution.

Experimef/t II

SJay green assessment:

13eginning from two days aller the commencement of this study, decline in physiological activities wereobserved in both varieties as their leaves began to wiLher. The withering began from the older leaves whichprogressively turned /Tom the normal green colour to brown and drying from the tip of each leaf.

Leaf reJcnJiofl capacity:

The result of the leaf retention capacity of the two varieties (Fig.~ 1) revealed a rapid decline in thenumber of leaves as they withered and died prematurely especially in the control and in O.006M H2SO]

respectively. However, var. LSr-047 exhibited a greater tolerance capacity tlianvar. CSr-054 especially atthe lower concentration (O.004M) of H1SOj as the two inner leaves [survived up till. the] 5th day in thismedium. i'

Progressive development of flowering phase:

In sugarcane, four distinct phases cf flowering is recognizable and tbe sequence is as follows:

initiation ~ flagging ~ tipping ~ arrow emergence. Although the two varieties were at the initiationphase when the experiment was set up, neither of the two could proceed to the next phase even at the lowerconcentration of O.004M (H2SOj) and this may be due to disruption in the hormonal system of the plantsresulting from toxicity of the solution. .

504

G----€> LSI-054 CONTROL

I I C06808 CONTROL

[]---El LSI-054(0.0021\:f)

~ C06808!(0.002M)

1!.----A LSI-054(0.006M)

.---JE C06808(0.006M)

9 11 13 15 17 19

Days

Fi~. 2a Leaf retenlion capacity ~ithe flowering stalks of two sugarcane "aiieties immersed illdifferent concentrations of

preservative medium H,So.·

8Ul Q);>(";jQ)....:I 6l:: Q)Q)~CO'-H00-;L

19171513

o 0 LSI-054 CO}JTROL

I I LSI-047 CO}JTROL

o D LSI-054(0.004M}

o <> LSI-047 (0.004M)

.A A LSI-054(O.006M)

W W LSI-047(O.006M)

53 11

DaysFig. I Leafretenlion capacity of the !lowering stalks of two sugar

cane varieties immersed in ditTerent concentrations of

preservative l11ediuOl H:SOJ

oZ

10

UlQ);>

~ 6>-l

~Q)~bJ)4

'-Ho

U1aU1

.~.,..

F..xperimellf II

Slay green m:\'essmellf:

Generally, canes at the vegetative phase were more tolerant to the different concentrations or the dil.H2S04 as well as the control than those at the flowering phase. This high level of tolerance exhibited by

the young canes may be due to higher accumulation of growth factors in the young canes than in theflowering ones or that there is less demand for these essential nutrients during the vegetative phase.

However, the trend in the decline in physiological activity as indicated by withering and subsequent deathof leaves was similar to what was observed in experiment 1 regardless of the age of the canes.

Leqlrelelllioll cupacity:

Leaf retention capability of the young canes in the different concentrations of H2S04 and in the controlare presented in Table J. For this parameter, attention was focused only on the four innermost leaves !IS

these were the youngest and most physiologically active. All the test varieties responded similarly in the

control and the only remarkable difference' in varietal response at!,his stage was at a concentration of0.002M H2S04 where var. LSI-047 appeared to be more tolerant than'the others. For example, while thetwo innermost leaves remained alive until the experiment was terminated (51 days), those of vars. LS.I-050

and C06806 were alive for only 35 and 41 days respectively. The response of the cane stalks to the othertwo concentrations did not differ much even though the varieties' changed ranking in their leaf retentioncapability (Table I).

Table I:Leaf retention capacity of young (vegetative phase)sugarcane stalksimmersedIndifferent

concentrations of weak acid (diL H:,SO'I)

Variety

NooCdays for which leaf remained physiologicafly active

Leaf No.

O.OOM0.002M0.004M0.006M

LSI-047

35+514549

2

35514045

3

30202535

4

15152020

LSI-050

35355050

2

35354550

3

30304040

4

20204040

C06806

35355045

2

35414545

3

2040'40 40

4

5253020

+ = Values are the means of two replicates.

506

The rate in loss of physiological activity was taster at the flowering phase that at the vegetative phaseregardless of the concentration of the preservative medium (Fig. 2) Both vars. LSJ-054 ancl (06806showed differential response to O.002M and O.006M res~ectively with the former exhibiting higher

tolerance capacity at O.006M H2S04 while the latter exhibited similar response to both concentrations (Fig.2a). Val'. 136609 on the other hand was intolerant of either cqncentration (Fig. 2b). Comparison among thefour varieties in their tolerance to O.006M lhSO~ (Fig. 3) revealed that vars. LSI-050 and LS1-054 bothexhibited greater tolerance level than the other two varieties as they survived longer than the other twovarieties (app. 8 days).

Similar to what was observed in experiment r, none of the test varieties could proceed to the next

flowering phase (i.e. the flag stage) irrespective of the concentration of the preserval ivc mediull1 usedHowever, there were initiation of rooting as well as emergence of side shoot both at the vegetative and

flowering phases (Plates 1. 2 and 3). At the vegetative phase, rooting occurred in var. LSI-047 beginningfrom the fifth day in both the lowest and highest concentrations (Plate I) but not in the intermediateconcentration while emergence of side shoots were observed beginning from the 7Ul day at the Slh node in

this medium. While neither rooling nor side shoot emergence occurred in-var. C06806, var. LSJ-OSD rooted

only in the highe'1>tconcentration. The root~ de\1elopeO. and progre:>:>i\1elyhardeneO after 32 lia)':> and laterdied. The two side shoots produced by var. LSf-047 (Phase r) measured 15 and- 25cm respectively andwere alive until the experiment was terminated.

At the flowering phase, rooting and emergence of side shoots occurred in two of the test varieties (LSl­

050 and C06806) immersed in the medium containing 0.002M H2S04 (Plates 2 and 3). Root initiation wasobserved also as from the fifth day in the two varieties and this was followed by emergence of side shootsas from the 7111 day beginning from the sixth node. Root proliferation was faster and higher in var. LSI-050than in var. C06806 (plate 2). By the lih day, three mare. node~ \llZ:. 71h, gU, and 9"1 (arrowed> lat.erproduced side shoots (Plate 3) which developed capidly than the older shoots suggesting apical d(lmirJ8.flcc

in nutr·lent sourc·lIlg. Follow·lIlg root initiation and side shoot emergence, tile solut"ton .1Ilthe jars reduced ata faster rate in these varieties compared to others which neither rooted nor produce side shoots. This isprobably due to increased metabolic activities in the stalks. Drying of the side shoots was observed from52 days after the experiment was set up.

Discussion

The primary objective of this study was to formulate an appropriate preservative medium which could

keep cut surgarcane stalks in a viable state during the period of hybridization and fuzz ripening. This

period is expected to last up to three weeks. The results obtained in this study revealed that test varieties

survived in the formulations from H2S04 for more than the expected-period. In other words, even at thesame molarity, formulations tram H2SO,\ served as better preservative media than formulations of H2S03.

For example, cane stalks at both the vegetative and flowering phases responded better by (i) beingphysiologically active for a longer period and (ii) producing roots and side shoots in the differentconcentrations of the dilute H2S04• while they could only survive for between 5 and 9 days in those ofH2S03 and without root initiation nor side shoot emergence (Figs. 2 and 3, Plates 1;2 and 3)

In sugarcane, development of side shoots is a common feat~re in flowering varieties especially after thedeath of the main stalk following fuzz ripening. Thus the occurrence of side shoots in these varietiesindicated that the stalks were still physiologically active despite the initial withering of the leaves.However, while the initiation of rooting and/or emergence of side shoot suggests stability of the ·stalks· inthe preservative solution after the initial shock, it was apparent that the disruption in the physiologicalactivities had terminated the flowering process. Consequently and upon recovery from the initial shock, the

hormones responsible for rooting and side shoots in the respective primordial wer;;- probably activatedresulting in rooting and side shoots observed. This implies that the two events (flowering versusrooting/side shoot emergence) are independent of the otherknd are probably controlled by differenthormones. The ability of H2S04 to act as a rooting hormone has also b~n reported by Jones a.nd Hatfield(1976).

507

G------€) L51:050

I I L51-054

~B6609

~C06806

2

8

5 ,7 9 11 13 15 17 ' 19

DaysFig, 3 Leaf retention capacity of the !lowering stalks of four sugar

cane varieties immersed in 0.006M H,SO,

10

C<lJ<lJI-bll

4­ooZ

VJ<lJ

>cj<lJ

..J

G-----E) L51-054 CONTROL

I I C06808 CONTROL

[}--{l LSI-054(0.002M)

~ C06808 (O,002M)

A-------A LSI-054(O.006M)

3 5 11 13 15 17 19

Daysfig, 3 Leaf retention capacity of the flowering stalks of four sugar

cane varieties immersed in 0.006M H,SO.

10

8

~ 6>:\::J

J.-l

..:::J:JI­~

'--<o0'2ZU'1o

(Xl

2 3 4 5 6

Pi,di:: I. Phc)tograph showing the extent of root formation in young stalk~ of twosllgcrCl.1ne varieties immersed in three different COllcelltrations of the preservativenlcdiul1i. Note that var. LS-047 failed to root in imcrmcdia1c conccntnitioLJ bell rooted in

10\ver "IlI(J higher com;entralions.

(I) f..S-050, 0.0021\/1 H2S04; (2) LS-047, O.OOLM H2SO.\; (J) LS-OSO, O.004M H2S04;

(4) LS-047, O.004M H2S04; (5) LS-050, 0.006'\'1 H2S0-1; *6) LS-047:0.006M H2S04,

508

Fig. 2: Photograph showing the extent of root proliferation in the following stalks ofvar. CO-6806 and LS-050 immersed in a.a02M H2S04 after the death gy,eenleaves. The dark colouration in roots of LS-050 showed that the roots were already old.

510

!", ,'.",

Fig. 3: Photograph showing the emergence of side shoots in flowering stalks of vars.CO-6806 and LS immersed in O.002M H2S04 after death of the green leaves.

511

The local (LSI-) varieties appeared to have a higher tolerance level than the exotics This isexemplified by the high sensitivity ofvar. B6609 as compared to var LSI-050 (Fig 2b) and the extent ofroot proliferation in var LSI-050 relative to that of var. C06806 (Plate 1). Similar differences intolerance to preservative solutions have been reported by earlier workers For example, Bourne (1929)noted that the Florida first nobilizations of"Chunee" variety kept better in the solution than the noblecanes while Warner (1953) also reported that some of the Barbados varieties could not tolerate theconditions in solution as well as most of the locally bred varieties

Our results especially for the H2S03 is contrary to the success previously reported in Hawaii(Warner. 1953) However. Stevenson (1965) noted that in spite of its property to maintain growth,flowering and seed setting in cut canes, H2S03 is well known to be toxic to cane tissues when inconcentrations above the minimum required by the plants and that its function is not really wellunderstood. This tends to suggest the need to find means of regulating the absorption of the acidsolutiOn Walker (1960) and Stevenson (1965) have suggested certain modifications when usingsulphurous acid as preservative medium to cut canes These include coating leaf-sheaths of cut caneswith latex emuision to prevent stalks from absorbing quantities of the acid solution more than theminimum required and cutting the surface of the stalk at least once at the time of renewal of theSQlution. Therefore, it may be necessary to conduct further studies, aimed at regulating the absorptionof the stock solution from these formulations or lower their concentrations (for example, ofthe order ofID") as a means of eliminating their toxic effects Alternatively, the bc:'.<lviour of new substances suchas Na2 S03, CH3 COOH which (may eliminate the toxic effect ofH+) could be investigated.

ReferencesBourne (1929) The evolution of a philosophy of sugarcane breeding in Hawaii. Hawaiian PlantersRec.54.N03.pp. 139-162.Hem/, DJ. (ed.) (1987). Sugarcane improvement through breeding. Amsterdam, New York. Elsene/,USA.

Jones, 0.0 and S.G.S. Hart field (1976). Root initiation inApple Shoots cultured in vitro with Auxj nsand Phenolic compounds. Jour. HoL Sc. 51: 495 - 499.Olaoye. G. (1996). Genetic variability between and within progenies of sugarcane cross developed bymodified

polycross method at the seedling selection stage. Proc 14,bAnnual Conf. HO RTSON: 231-237Stevenson. G.c. (1965). Genetics and Breeding of sugarcane. 272pp.Walker. D.I T. (1960). Useful information from variety trials. Proc. B w.,T. Sugar Tech. Meeting, pp.312-318.

Warner. IN (1953). The evolution of a philosophy on sugarcane breeding in Hawaii. HawaiianPlanters Rec. 54. No.3.pp.139-152.

512