Malaysian Cocoa Journal 1

Malaysian Cocoa Journal 2

MALAYSIAN COCOA JOURNAL

ADVISER

Dato’ Dr. Azhar Ismail

CHIEF EDITOR

Dr. Rosmin Kasran

ASSISTANT CHIEF EDITOR

Dr. Lee Choon Hui

SECRETARY

Dr. Tan Chia Lock

EDITORIAL COMMITTEE

Denamany Gangadaran Alias Awang Dr. Douglas Furtek Suzannah Sharif Hii Ching Lik Datin Norhaini Udin Hj. Omar Hj. Tompang Ramle Kasin

Published by Malaysian Cocoa Board, 5th & 6th Floor, Wisma SEDCO, Lorong Plaza Wawasan, Off Coastal Highway, 88999 Kota Kinabalu, Sabah, Malaysia

Malaysian Cocoa Journal 3

©All rights reserved. No part of this publication may be reproduced in any form or by any means without permission in writing from Malaysian Cocoa Board

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TABLE OF CONTENTS FORUM PERSPECTIVE FOR COCOA CULTIVATION IN MALAYSIA : RE-LOOK AT THE 1 ECONOMIC INDICATORS Azhar I. and M. T. Lee

BIOTECHNOLOGY EXTRACTION OF DNA FROM COCOA (THEOBROMA CACAO L.) 19 Lea J.

COCOA GENETIC LINKAGE MAP CONSTRUCTION TOWARDS QTL MAPPING 24 Lea J., K. Lamin and D. B. Furtek

RECURRENT EMBRYOGENESIS AND IMPLICATIONS FOR GENE TRANSFER IN 28 THEOBROMA CACAO L. Tan C. L. and D. B. Furtek

SOMATIC EMBRYO GERMINATION AND CONVERSION INTO PLANTLETS IN COCOA 36 (THEOBROMA CACAO L.) Tan C.L.

ENTOMOLOGY DISPERSAL OF TRICHOGRAMMATOIDEA BACTRAE FUMATA NAGARAJA 40 (HYMENOPTERA: TRICHOGRAMMATIDAE) AFTER RELEASES IN A MALAYSIAN COCOA FIELD Azhar I. and G. E. Long

EFFICACY OF MATING DISRUPTION USING SYNTHETIC SEX PHEROMONE FOR THE 46 MANAGEMENT OF COCOA POD BORER, CONOPOMORPHA CRAMERELLA (SNELLEN) (LEPIDOPTERA: GRACILLARIIDAE). Alias A., W. Sadao and E.B. Tay

CHEMISTRY- PROCESSING AND PRODUCT DEVELOPMENT QUALITY ASSESMENT OF COCOA BEANS PRODUCED BY SMALLHOLDERS FROM 53 DIFFERENT REGIONS IN MALAYSIA Hii C. L., Y. K. C. Samuel and I. Nor Haslita

D-FRUCTOSE ADDITION DURING MALAYSIAN COCOA NIBS ROASTING 59 Suzannah S.

EFFECTS OF BLENDING COCOA BUTTER AND CAROTINO OIL ON THE PHYSICAL 67 AND SENSORY QUALITIES OF FAT BLENDS AND CHOCOLATE Wan Aidah W.I., H. Asimah, B. Abd. Salam and S. Mamot

DETERMINATION OF HYDRO DISTILLED ESSENTIAL OILS IN THEOBROMA CACAO L. 74 Samuel Y. K. C., A. M. Sri Nurestri and R. Nazarudin

SHORT COMMUNICATION TAXONOMIC STATUS OF TRICHOGRAMMATOIDEA BACTRAE FUMATA NAGARAJA, AN 83 EGG PARASITOID OF THE COCOA POD BORER, CONOPOMORPHA CRAMERELLA (SNELLEN) IN MALAYSIA : A CRITICAL REVIEW Alias A., I. Azhar and M. Schilthuizen

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NEW RECORD OF TRICHOGRAMMA CHILONIS ISHII (HYMENOPTERA: 86 TRICHOGRAMMATIDAE) AS AN EGG PARASITOID OF THE COCOA POD BORER, CONOPOMORPHA CRAMERELLA (SNELLEN) (LEPIDOPTERA: GRACILLARIIDAE) IN MALAYSIA Alias A., M. Schilthuizen and H. Sulaiman

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Forum PERSPECTIVE FOR COCOA CULTIVATION IN MALAYSIA : RE-LOOK AT THE ECONOMIC INDICATORS

Azhar I. and M. T. Lee Malaysian Cocoa Board, Locked Bag 211, 88999 Kota Kinabalu, Sabah, Malaysia.

Malaysian Cocoa J. 1: 1-18 (2004) ABSTRACT The various economic and related factors, which include cocoa prices, cocoa pod borer, labor, productivity, farm economics and competitiveness, were reviewed to come out with the economic indicators for assessment of the perspective for cocoa cultivation in Malaysia. The analysis shows that despite of relatively low prices of cocoa recorded in certain period in the past, the average price in the past 30 years was US$1,806/t or RM4,712/t, which is considered high and very attractive for investment. The large variation observed in the prices of cocoa provides the opportunity for profit taking in investment. With appropriate application of available technology, cocoa pod borer can be effectively managed with little crop loss through sound management. With a good understanding of the crop requirement and appropriate application of available technology, the labor requirement in cocoa cultivation can be minimized and efficiently managed. There is clear indication of excellent potential for improvement in productivity from the Malaysian national average of about 0.9 t/ha to 2 t/ha or higher through appropriate utilization of planting technology and sound management with commitment and care as the key success factors in cocoa cultivation. At a productivity of 1.5 t/ha or higher, there is always profit in return on investment. The annual rate of return on investment (RROI) can be very attractive at a rate of up to several hundred percent or higher depending on the levels of productivity and prices. It was indicated that a family of smallholders can look after up to 8 ha of cocoa farm without the need of employing hired labor. Reasonable to high level of income for a family can be derived from cocoa cultivation with a farm size of between 6 and 8 ha at a productivity of 1.5 t/ha or higher. The cocoa planting industry in Malaysia has the competitive edge in many aspects and will have to be driven by technology and management push for high productivity and efficiency to ensure the competitive edge. On the basis of the above analyses, it is concluded that cocoa cultivation is very much an economically viable investment in contributing to the national economic development in Malaysia.

Key words: Malaysia, planting industry, cocoa

INTRODUCTION

Under the crop diversification program of the National Agricultural Policy, cocoa (Theobroma cacao L.) was introduced into Malaysia (then Malaya, North Borneo and Sarawak) for commercial planting in 1950s (MCB, 1991). The cocoa industry grew to become the third major commodity crop in Malaysia after oil palm and rubber. However, after reaching a peak of 414,236 ha in area in 1989 and 247,000 tones dried cocoa bean production in 1990, the cocoa planting industry declined to 45,365 ha and 36,236 t dried cocoa beans in 2003 (Figure 1). Because of the shifting of the major plantation houses from cocoa for oil palm planting, smallholder sector has become the dominant player in cocoa cultivation with an area of 29,951 ha as compared to 15,415 ha under estate sector in 2003 (MCB, 2004a).

The growth of the cocoa planting industry has led to the establishment of the cocoa downstream industry in Malaysia in 1973 and its rapid development to attain an annual grindings of 167,595 t with a grindings capacity of 260,000 t in 2003 (Table 1). The cocoa grindings industry in Malaysia is expected to grow further to 400,000 t in the near future.

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('000 ha or tonnes)

Area (ha) Production (tonne) 450

400

350

300

250

200

150

100

50

0 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003

Figure 1. Trend of cocoa planting area and dry cocoa bean production in Malaysia (Anon, 1992, 2003; Department of statistics, Malaysia, 2004).

Table 1. Malaysian grindings of cocoa beans.

Year Grindings (tonnes)

1980 6,000 1985 27,000 1990 70,000 1995 103,540 2000 139,443 2003 167,595 Sources : Malaysian Cocoa Board (MCB, 1992 ; 2004).

The increasing demand of cocoa beans for local grindings and the declining local cocoa bean production has turned Malaysia from previously a net exporter of cocoa beans to be a net importer of cocoa beans since 1997. Consequently, the situation resulted in significant outflow of foreign exchange amounting to RM1.08 billion for the import of 234,776 t of cocoa beans in 2003 (Table 2).

The declining trend of cocoa planting in Malaysia gave the general impression of little prospect for cocoa cultivation. Economic and related factors, such as low prices, cocoa pod borer infestation and labor constraints, are often quoted as the reasons for moving out of cocoa cultivation. However, one would wonder whether the apparent negative view on the fate of cocoa cultivation and its possible contribution to the national economic development is correctly drawn.

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It is of interest to note that while Malaysia is experiencing a downtrend in cocoa cultivation, other cocoa producing countries in the world, namely West Africa, Latin America and Asia, are either maintaining or expanding the growth of their cocoa planting industry (ICCO, 2004). Furthermore, feedbacks from the industry indicated that well-managed cocoa holdings in Malaysia were able to sustain profitable over the years of fluctuating prices. These observations suggest otherwise that cocoa cultivation can be an economically viable investment. It rings the bell for the need of a closer look at the various economic and related indicators to clear the air on the economic perspective for cocoa cultivation. This paper is to give an overall analysis on the various economic and related factors and to come out with the respective indicators for the assessment of the prospect for cocoa cultivation in Malaysia. The economic and related factors to be addressed include cocoa prices, cocoa pod borer, labor, productivity, farm economics and competitiveness.

Table 2. Malaysian export and import volume and value of cocoa beans.

Export Import Year Volume (tonnes) Value (RM’000) Volume (tonnes) Value (RM’000)

1985 81,465 409,459 50 240

1990 162,618 448,452 101 123

1995 52,533 171,981 39,704 123,737

1997 30,960 114,114 37,127 145,324

2000 11,408 32,838 106,701 292,410

2003 13,061 82,458 234,776 1,076,554 Source : Malaysian Cocoa Board (MCB, 1992 ; 2000 ; 2004)

COCOA PRICES

As in any commodities, the price of cocoa beans is one of the most significant factors influencing the pace of development of the cocoa planting industry in Malaysia as well as in other parts of the world. High prices in 1970’s and 1980’s had led to unprecedented accelerated growth of the cocoa planting industry and the relatively low prices that followed were among the major factors that contributed to the decline of cocoa planting in Malaysia (Figure 1 and 2).

Highest and Lowest Prices – Figure 2 shows that world cocoa price experienced large fluctuation with extreme highs and lows in the past 30 years. Some of the indicators of the world cocoa prices during this period are presented in Table 3. The highest world daily price of cocoa beans was attained at US$4,723/t in July 1977 with the corresponding local price ex-Tawau up to as high as RM13,000/t. Whereas the lowest world daily prices of cocoa beans were recorded at US$912/t in June 1992 and US$774/t in November 2000 with the local prices of RM1,800/t and RM2,500/t respectively (Table 3). This represents a ratio of highest to lowest cocoa prices at 5.2 to 6.1 in US dollar or at 5.2 to 7.2 in Ringgit Malaysia, which is considered the highest among the commodity crops.

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Table 3. Indicators of world cocoa prices during the 30 year period from 1972/73 to 2001/02.

Prices of dried cocoa beans Indicators US$/t RM/t Date/Duration

Highest daily price 4,723 13,000* 18th July 1997

Lowest daily prices a) 912 1,800* June 1992 b) 774 2,500* 24th November 2000

a) 2,195 5,235** 1972/73 to1987/88 Annual average daily prices b) 1,306 3,411** 1988/89 to 1996/97 c) 1,300 4,956** 1997/98 to 2001/02

30 year annual average daily 1,806 4,712** 1972/73 to 2001/02 price Sources : ICCO (1992, 1997, 2000, 2004) * Prices ex-Tawau, Malaysia ** Annual average currency exchange rates of RM to US$ are used in computation.

US$/tonne

4000 70

3500 60 3000 50 2500 40 2000 30 1500 20 1000

500 10

0 0 61 66 71 76 81 86 91 96 00 02 1960/ 1965/ 1970/ 1975/ 1980/ 1985/ 1990/ 1995/ 1999/ 2001/ Price (US$) Stocks to grindings ratio

Figure 2. World cocoa price trend and stocks to grindings ratio (ICCO, 2003).

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Large variation in the prices of cocoa can be considered as a risk as well as an opportunity for profit-taking. For the cocoa growers who are able to reduce the cost of production to less than the anticipated lowest levels of cocoa price at any given time, there is always profit to make even at low prices and the opportunity to reap handsome return at times of high prices. In view of this, the anticipated lowest levels of cocoa price at any given time can be a useful reference point as the upper limit of production cost that ensure profit making at all times. Figure 3 shows a positive trend of historical lowest annual average daily cocoa prices over the past 40 years. Should this time trend of lowest world cocoa prices hold true, the future lowest levels of world cocoa prices are expected to be no less than US$1,000 in the next 5-10 years.

Average Daily Prices - High prices of cocoa beans had extended over a long period of 16 years from 1972/73 to 1987/88 with an average of US$2,195/t or RM5,235/t (Figure 2, Table 3). The cocoa growers had enjoyed extreme high profit during this period. The prices of cocoa came down thereafter and the annual average daily prices since 1988/89 was at about US$1,300/t or an equivalence of RM3,411/t for the period between 1988/89 and 1996/97, and RM4,956/t for the period from 1997/98 to 2001/02, respectively. Higher average daily prices observed in the more recent period were due to the result of a weaker Ringgit Malaysia that was pegged against the US dollar. Despite of being lower than that in the previous peak period, these levels of annual average prices of cocoa beans remain attractive and profitable to many cocoa growers. In addition, the annual average price of cocoa beans for the past 30 years from 1972/73 to 2001/02 was at US$1,806/t or an equivalence of RM4,712/t, which is considered very attractive for investment.

Supply, Demand, Stocks to Grindings Ratio and Prices - World cocoa price is influenced by the market force of demand and supply, and the stocks position. Changes in the annual position of supply and demand of cocoa beans and thus the year end stocks and the stocks to grindings ratio determine the direction of price movement. Figure 4 shows that the world annual production and consumption of cocoa beans increased from about 1.0 million tones in 1960/61 to 3.1 million tones in 2002/03 at an annual growth rate of about 2 - 3%.

Stocks to grindings ratio are considered as a good indicator of world cocoa prices and its movement. Figures 2 and 5 show that the world cocoa price is inversely correlated to stocks to grindings ratio and thus a lower stocks to grindings ratio will lead to higher cocoa prices, while a higher stocks to grindings ratio will result in lower cocoa prices.

According to the International Cocoa Organization (ICCO), a cocoa price of between £1,000 and £1,100 per t is considered as reasonable remunerative return to the cocoa farmers (ICCO, 2003). In order to attain this level of remunerative cocoa price, the stocks to grindings ratio will have to be managed at around 34% or lower Furthermore, it is the common objective of the international cocoa bodies, namely the ICCO and Cocoa Producers’ Alliance (CPA) to ensure sustainable remunerative cocoa prices to the farmers under the program of sustainable cocoa economy in the new International Cocoa Agreement 2001. With concerted efforts through international cooperation, there is a good possibility of successfully regulating the supply and demand and thus achieving the objective of maintaining good prices for the cocoa growers, as well as producing adequate volume of cocoa beans to meet the consumption requirement.

COCOA POD BORER

The cocoa pod borer or CPB (Conopomorpha cramerella (Snell)) is a pest of concern to the cocoa growers ever since its first discovery in Malaysia in 1980. CPB has been singled out as one of the major factors that dampened the interest expressed in cocoa cultivation because of the possible crop loss due to the pest and the need of labor and cost for its management. However, with appropriate application of available technology that includes the use of cultural practices such as sleeving, chemical treatments and other measures, CPB can be effectively kept under control with little crop loss and the labor requirement and cost can be kept to the minimum through accountable implementation of integrated pest management (IPM).

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1200 Crop year Price (US$/t) 1964/65 389 1971/72 583 1000 1992/93 1,051 1999/00 919

800

600 Adj R Square (power model)= 0.9141* Price (US$/t) = 374.068209*(Num_yr**(0.267412))

400

Annual average ICCO daily cocoa price (US$/t) 200 1960/61 1964/65 1969/70 1974/75 1979/80 1984/85 1989/90 1994/95 1999/00 2004/05 2009/10

Crop year

Figure 3. Time trend of historical lowest annual average ICCO daily cocoa price from 1960/61 to 2001/02 (ICCO, 2003).

‘000 tonnes 3500 3000 2500 2000 1500 1000 500 0 -500 1960/61 1970/71 1980/81 1990/91 2000/01

Surplus/Deficit Consumption Stock Production

Figure 4. World cocoa bean production, consumption and stocks (ICCO, 2003).

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$4,500 ) t /

$ $4,000 Adjusted R square = 0.66181** S Price (US$/t) = 6384.883612 - 165.184206*(Ratio) + 1.316974*(Ratio**2) U

( with 95% confidence interval e $3,500 c i r p a

o $3,000 c o c

y

l $2,500 i a d

O $2,000 C C I $1,500 age er

av $1,000

l nua

n $500 A

$0 25 30 35 40 45 50 55 60 65 70 Stocks to grindings ratio (%)

Figure 5. Relationship between stocks to grindings ratio and annual average ICCO daily cocoa price from 1977/78 to 2001/02 (ICCO, 2003).

Furthermore, the on-going R & D activities of the Malaysian Cocoa Board indicated the potential for the development of more cost effective labor saving technology for the control of CPB through the use of biological control agents and resistant planting materials as the main strategies for CPB management (Azhar, 1995, Azhar et al., 2000). With the development of these new technologies and their application, substantial reduction in labor requirement and CPB management cost can be anticipated.

LABOUR

The expenditure on labour is a major component of the production cost in cocoa cultivation. The requirement of labour for the maintenance of mature cocoa fields can be very variable from case to case depending on a number of factors which include farm terrain, tree height, pest and disease incidence, weed growth, productivity and others (Lass, 1985). The standard and quality of labour management can play a significant role in the efficiency of labour usage.

Our experience shows that with a good understanding on the crop requirement and the available technology for crop management, the labour requirement for carrying out various field tasks or activities can be more efficiently utilized and significantly reduced. For example, as there should be little or no weed growth in the cocoa fields with healthy plant growth and complete closure of canopy, the need of labour for in-field weed control can be negligible, except at the field perimeter. As shade removal is now commonly practiced for high productivity in mature cocoa, no labour is required for shade maintenance. Whereas, the

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labour requirement for harvesting, pest and disease management, pruning and others can be substantially reduced, if the height of cocoa tree is kept low within easy reach of the workers. If terrain allows, mechanization can be organized to reduce labour requirement for fertilizer application, pest and disease management and in-field transportation (Taylor et. al, 1982; Lim et. al, 1987; Chong et. al, 1990; Ho et. al, 1990; Chong and Tan, 1994). Mechanical tree pruner can be used for pruning of cocoa trees to reduce labour requirement (Hidayatullah and Hii, 1998). Furthermore, harvesting of cocoa is usually on contract basis. As it is a relatively light job as compared to harvesting of oil palm fresh fruit bunches, family hands of both sexes and all age groups can assist in harvesting cocoa pods to earn additional income for the family. The use of family hands for harvesting could be appropriately organized and capitalized to ease the pressure on labour requirement. Furthermore, the technical capability of the field workers could be strengthened to enhance efficiency through training with incentives as the driving force.

In taking into considerations of the above and in addition to the published information as well as our personal communication with the cocoa growers on labor usage, the labor requirement for field maintenance can be reduced to 36 man-days/ha/annum or at a ratio of 8 ha to one worker (Table 4).

Table 4. An example of labour requirement for maintenance and harvesting and pod breaking in mature cocoa fields at different levels of productivity.

Task Labour required (man-days/ha/annum) 1 t/ha 1.5 t/ha 2.0 t/ha 2.5 t/ha 3 t/ha

Fertilizer application 4 4 4 4 4 Weed control 4 4 4 4 4 Field Pruning 8 8 8 8 8 maintenance Pest and disease 20 20 20 20 20 management Sub total 36 36 36 36 36 (8 ha)* (8 ha)* (8 ha)* (8 ha)* (8 ha)*

Harvesting and pod breaking 15 23 30 38 45 (19.2)* (12.5)* (9.6)* (7.6)* (6.4)* Total 51 59 66 74 81 (5.6 ha)* (4.9 ha)* (4.4 ha)* (3.9 ha)* (3.6 ha)* * The figure in brackets indicates the ratio of area in hectares to labour on a total of 288 working man- days per annum.

Whereas, 15 man-days are required for harvesting and pod breaking in the production of one t of dried cocoa beans. This represents a requirement of 51 to 81 man-days/ha/annum or at a ratio of between 5.6 ha and 3.6 ha to a worker respectively for field maintenance, harvesting and pod breaking in cocoa cultivation with a productivity of 1.0 to 3.0 t/ha. As indicated above, the labor requirement in cocoa cultivation is relatively lower than commonly quoted. The relatively higher labor requirement in cocoa cultivation as compared to oil palm cultivation is largely due to the need of larger percentage of labor for pest and disease management, namely the CPB. Successful development of labor saving technology for CPB management will narrow the gap in labor requirement comparable to that in oil palm cultivation.

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PRODUCTIVITY

Productivity is one of the most important economic factors affecting the cost of production per tonne of cocoa beans. At higher productivity, the cost of production per t of cocoa will be lower and vise versa. In view of this, effort should be made to increase the productivity to as high as possible to maximize profit making.

Table 5 shows that the Malaysian average national productivity in cocoa cultivation increased from 625 kg/ha in 1990 to 932 kg/ha in 2002 and 799 kg/ha in 2003 (MCB, 2004a). At these levels of average national productivity, Malaysia is considered as the most productive cocoa producer in the world (ICCO, 2004). However, these levels of average national productivity are far lower than the theoretical potential yield of 11 t/ha (Corley, 1967) and the attainable yields of between 2.0 and 6.8 t/ha (Lee and Chong, 1987; Mohd Yusof et. al, 1998). Furthermore, the smallholder cocoa holdings under the rehabilitation program implemented by the Malaysian Cocoa Board have attained significant yield improvement from less than 0.5 t/ha to up to 4.0 t/ha with an average of around 2.0 t/ha (MCB. 2001, 2004). Excellent potential for productivity improvement is clearly indicated.

Table 5. Some indicators of productivity in cocoa cultivation.

Indicator Productivity (t/ha) References

Malaysian national average productivity : 1990 0.628 MCB (2004a) 1995 0.692 2000 0.927 2002 0.932 2003 0.799

Theoretical potential yield 11 Corley (1967)

Highest yields recorded in a 5.0 to 6.8 Lee and Chong (unpublished) year Lee and Chong (1987) Average yields of well- 2.0 to 4.6 Mohd. Yusof et. al. (1998) managed cocoa farms

Feedbacks from well-managed cocoa holdings and the experience of the smallholder rehabilitation program indicate that with the utilization of the available planting materials and planting technology coupled with sound management, a yield of 2 to 3 t/ha can be within easy reach of the cocoa farmers. It was indicated that a good understanding on the crop requirement and the available planting technology and their adoption coupled with “commitment and care” in management are the key factors for success in achieving high productivity (Lee and Chong, 1987; Mohd Yusof et al., 1998). Taking into consideration of the importance of these requirements for attaining high productivity, “Accountable Management System” (AMS) is suggested for adoption cocoa cultivation (Lee and Azhar, 2004). In the AMS as suggested, the requirement of “commitment and care” are incorporated into the system for management in the form of “responsibility” and “trace-ability” that allow all subject matters, activities and personnel are accountable collectively leading towards the attainment of not only high productivity, but also high efficiency and quality in cocoa cultivation. AMS is operated as a system equivalent to the acquisition of the services of committed highly successful cocoa farmers or growers for management to attain high productivity and efficiency among others in cocoa cultivation.

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FARM ECONOMICS

For the assessment of farm economics, the cost of production, income from the sale of dried cocoa beans and thus the net farm return of investment and the rate of return on investment (RROI) will be considered. In addition, the income of cocoa smallholders in relation to farm size per holding will also be discussed.

Production Cost - Table 6 presents an example of the unit cost of production components including input, labor for field maintenance, harvesting and pod breaking activities, and overheads and general charges in cocoa cultivation. Whereas, the estimated production costs in estate-scale cocoa holdings and smallholder cocoa farms using hired labor or own labor at a productivity of between 1 and 3 t/ha are given in Table 7.

Table 6. Estimated annual production cost by components in mature cocoa fields.

Production Cost Components Unit Cost*

A. Input (RM/ha) 1,000 (Fertilizers, chemicals, etc)

B. Labour for field maintenance (RM/ha) 900

C. Harvesting and post harvesting (RM/t) 500 (Labour and materials for harvesting, fermentation and drying of cocoa beans)

D. Overheads and general charges (RM/ha) 500 * The cost figures may vary from case to case.

Table 7 shows that in the estate-scale cocoa holdings, the cost of production increases from RM2,900/ha to RM3,900/ha at a productivity of 1 to 3 t/ha respectively, but it decreases from RM2,900/t at a productivity of 1 t/ha to RM1,300/t at 3 t/ha. The effectiveness of higher productivity in reducing the production cost per tonne of dried cocoa beans is clearly demonstrated. The costs of production in smallholder cocoa farms using hired labor or own labor are relatively lower as compared to that in the estate-scale cocoa holdings at the same level of productivity largely due to the exclusion of the overheads and/or labor costs in the assessment of the cost of production. However, with the overheads and general charges incurred in the management of estate-scale cocoa holdings, the productivity and efficiency are expected to be higher than the smallholder cocoa farms and thus a lower cost of production per t of dried cocoa beans is anticipated.

Farm Return and RROI - The estimated net farm return, which represents the profit or loss in income, and the rate of return on investment (RROI) for the estate-scale cocoa holdings and smallholder cocoa farms using hired labor or using own labor at a farm productivity of 1 to 3 t/ha and a price of cocoa beans ranging from RM2,000/t to RM8,000/t are presented in Tables 8, 9 and 10, respectively. The levels of net farm return and RROI are significantly influenced by the changes in the levels of cocoa price and the farm productivity.

Table 8 shows that at a cocoa price of RM2,000/t, the estate-scale cocoa holdings will have to have a productivity of 1.6 t/ha in order to break even for its investment and there will be profit to make at a RROI of 18% and 54% at a productivity of 2 t/ha and 3 t/ha, respectively. There is always a profit in return at a price of RM3,000/t even at a productivity of 1 t/ha. With a productivity of 1.5 t/ha and 3 t/ha, the profit margin or RROI will be 91% and 208% respectively at a cocoa price of RM4,000/t and up to 281% and 515% in RROI at RM8,000/t. Whereas, the levels of net farm return and RROI in smallholder cocoa farms

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either using hired labor or using own labor are relatively higher than that in the estate-scale cocoa holdings at the respective levels of productivity (Tables 9 and 10).

Table 7. An example of estimated annual production cost in estate-scale cocoa holdings and smallholder cocoa farms using own labour or using hired labour at different levels of productivity.

Estimated annual production cost at different levels of productivity 1 t/ha 1.5 t/ha 2 t/ha 2.5 t/ha 3 t/ha

Estate-scale cocoa holdingsa (RM/ha) 2,900 3,150 3,400 3,650 3,900 (RM/t) 2,900 2,100 1,700 1,460 1,300

Smallholder cocoa farms using hired labour b (RM/ha) (RM/t) 2,400 2,650 2,900 3,150 3,400 2,400 1,767 1,450 1,260 1,133 Smallholder cocoa farms using own labour c (RM/ha) (RM/t) 1,000 1,050 1,100 1,150 1,200 1,000 700 550 460 400 a Cost item A, B, C and D in Table 6. b Cost item A, B and C in Table 6. c Cost item A in Table 6 plus additional material cost at productivity higher than 1 t/ha.

Table 8. Net farm return and annual rate of return on investment (RROI) in estate-scale cocoa holdings at different levels of cocoa price and productivity.

Cocoa prices Net farm return (RM/ha) and RROI (%) at different levels of productivity (RM/t)a 1 t/ha 1.5 t/ha 2 t/ha 2.5 t/ha 3 t/ha

2,000 -900* -150 600 1,350 2,100 (-31%)** (-5%) (18%) (37%) (54%)

3,000 100 1,350 2,600 3,850 5,100 (3%) (43%) (76%) (105%) (131%)

4,000 1,100 2,850 4,600 6,350 8,100 (38%) (91%) (135%) (174%) (208%)

6,000 3,100 5,850 8,600 11,350 14,100 (107%) (186%) (253%) (311%) (382%)

8,000 5,100 8,850 12,600 16,350 20,100 (176%) (281%) (371%) (448%) (515%) a The average cocoa price in the past 30 years was at no less than RM4,000/t. The prices had reached > RM8,000/t in 2002. The projected lowest annual cocoa prices may not be less than US$1,000/t or a local price of RM3,000/t (refer to Fugure 3). * Net farm return in RM/ha = productivity x cocoa price - annual production cost. (refer to Tables 6 and 7 and cocoa prices) ** Figure in brackets denotes RROI as (net farm return ÷ annual production cost) in %.

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Table 9. Net farm return and annual rate of return on investment (RROI) in smallholder cocoa farms using hired labour at different levels of cocoa price and productivity.

Cocoa prices Net farm return (RM/ha) and RROI (%) at different levels of productivity (RM/t)a 1 t/ha 1.5 t/ha 2 t/ha 2.5 t/ha 3 t/ha

2,000 -400* 350 1,100 1,850 2,600 (-17%)** (13%) (38%) (59%) (77%)

3,000 600 1,850 3,100 4,350 5,600 (25%) (70%) (107%) (138%) (165%)

4,000 1,600 3,350 5,100 6,850 8,600 (67%) (126%) (176%) (218%) (253%)

6,000 3,600 6,350 9,100 11,850 14,600 (150%) (240%) (314%) (376%) (429%)

8,000 5,600 9,350 13,100 16,850 20,600 (233%) (353%) (452%) (534%) (606%) a The average cocoa price in the past 30 years was at no less than RM4,000/t. The prices had reached > RM8,000/t in 2002. The projected lowest annual cocoa prices will not be less than US$1,000/t or a local price of no less than RM3,000/t (refer to Figure 3). * Net farm return in RM/ha = productivity x cocoa price – annual production cost. (refer to Tables 6 and 7 and cocoa prices) ** Figure in brackets denotes RROI as (net farm return ÷ annual production cost) in %.

Table 10. Net farm return and annual rate of return on investment (RROI) in smallholder cocoa farms using own labour at different levels of cocoa price and productivity.

Cocoa prices Net farm return (RM/ha) and RROI (%) at different levels of productivity (RM/t)a 1 t/ha 1.5 t/ha 2 t/ha 2.5 t/ha 3 t/ha

2,000 1,000* 1,950 2,900 3,850 4,800 (100%)** (186%) (264%) (335%) (400%)

3,000 2,000 3,450 4,900 6,350 7,800 (200%) (329%) (445%) (552%) (650%)

4,000 3,000 4,950 6,900 8,850 10,800 (300%) (471%) (627%) (770%) (900%)

6,000 5,000 7,950 10,900 13,850 16,800 (500%) (757%) (991%) (1,204%) (1,400%)

8,000 7,000 10,950 14,900 18,850 22,800 (700%) (1,043%) (1,355%) (1,639%) (1,900%) a The average cocoa price in the past 30 years was at no less than RM4,000/t. The prices had reached > RM8,000/t in 2002. The projected lowest annual cocoa prices may not be less than US$1,000/t or a local price of no less than RM3,000/t (refer to Figure 3). * Net farm return in RM/ha = productivity x cocoa price – annual production cost. (refer to Tables 6 and 7 and cocoa prices) ** Figure in brackets denotes RROI as (net farm return ÷ annual production cost) in %.

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The above analysis demonstrated clearly the importance of cocoa prices and the levels of productivity and their combined effect on the net farm return and RROI and thus the viability and competitiveness of cocoa cultivation. With a potential attainable RROI of up to several hundred percents or higher as indicated above (Tables 8, 9 and 10), there is sufficient economic incentive for the management of cocoa holdings to put in the required effort to increase the productivity to the attainable yield levels of 2 t/ha or higher. Apart from keeping the unit cost of production components as low as possible, the need of labor and cost as required for the control of pest and disease, namely the CPB as claimed should not be a limitation from the investment point of view.

Farm Size and Income of Cocoa Smallholders - As smallholders are now the dominant players in cocoa cultivation in Malaysia, the income of the cocoa smallholders is an important consideration particularly under the national policy on poverty eradication. Tables 11 and 12 show that the income of cocoa smallholders is determined by the levels of productivity and the size of cocoa farm per holding. With a holding size of one hectare per family and at a cocoa price of RM4,000/t, which was the average price in the past 30 years, the net income of cocoa smallholders either using hired labor or own labor is estimated at RM279 or RM413/month at a productivity of 1.5 t/ha, and increases to RM425 or RM575, RM571 or RM738 and RM717 or RM900/month at a productivity of 2, 2.5 and 3 t/ha, respectively.

Whereas, at a productivity of between 1.5 and 3.0 t/ha and a cocoa price of RM4,000/t, the cocoa smallholders who own 4, 6 and 8 ha of cocoa farms will earn a monthly net income of between RM1,117 and RM3,600 RM1,675 and RM5,400 and RM2,233 and RM7,200, respectively (Tables 11 and 12). In view of the facts that a family can look after up to between 6 and 8 ha of cocoa farm without the need of hiring labor (Table 4) and the levels of monthly net income can be in between RM2,475 and RM5,400 and up to between RM3,300 and RM7,200 at a productivity of 1.5 and 3.0 t/ha, respectively (Table 11), a farm size of between 6 and 8 ha can be considered as the economic farm size for smallholders.

Table 11. Net income of cocoa smallholders using own labour at different farm sizes per holding and different levels of productivity at a price of cocoa beans of RM4,000/t a.

Productivity Net income (RM/year and RM/month) at different farm sizes per holding (t/ha) 1 ha 4 ha 6 ha 8 ha b

1.0 3,000* 12,000 18,000 24,000 (250)** (1,000) (1,500) (2,000)

1.5 4,950 19,800 29,700 39,600 (413) (1,650) (2,475) (3,300)

2.0 6,900 27,600 41,400 55,200 (575) (2,300) (3,450) (4,600)

2.5 8,850 35,400 53,100 70,800 (738) (2,950) (4,425) (5,900)

3.0 10,800 43,200 64,800 86,400 (900) (3,600) (5,400) (7,200) a Estimated 30 year average local price of cocoa beans. b A family is able to look after up to 8 ha of cocoa farm (Table 4). * Net income in RM/year. ** Figure in brackets denotes net income in RM/month.

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Table 12. Net income of cocoa smallholders using hired labour at different farm sizes per holdings and different levels of productivity at a price of cocoa beans of RM4,000/t a.

Productivity Net income (RM/year and RM/month) at different farm sizes per holding (t/ha) 1 ha 4 ha 6 ha 8 ha

1.0 1,600* 6,400 9,600 12,800 (133)** (533) (800) (1,067)

1.5 3,350 13,400 20,100 26,800 (279) (1,117) (1,675) (2,233)

2.0 5,100 20,400 30,600 40,800 (425) (1,700) (2,550) (3,400)

2.5 6,850 27,400 41,100 54,800 (571) (2,283) (3,425) (4,567)

3.0 8,600 34,400 51,600 68,800 (717) (2,867) (4,300) (5,733) a Estimated 30 year average local price of cocoa beans. * Income in RM/year. ** Figure in brackets denotes net income in RM/month.

COMPETITIVENESS

Comparative to Other Cocoa Producing Countries - The competitiveness of Malaysia as compared to other cocoa producing countries can be viewed as follows:

1. With an average national productivity of 0.8-0.9 t/ha (MCB, 2004a) as compared to a national productivity of 0.2-0.6 t/ha in other cocoa producing countries (ICCO, 2004), Malaysia is regarded as the most productive cocoa producer in the world. 2. Malaysia has a long history and well-established plantation management experience for achieving high productivity, efficiency and quality in the cultivation of plantation crops including cocoa. 3. A strong R & D foundation with focused R & D activities was established for the development of the cocoa planting and downstream industry in Malaysia. With appropriate application of the available planting materials and technology developed through R & D coupled with the plantation management experience to achieve high productivity, efficiency and quality is the strength of Malaysian cocoa industry. 4. Malaysia has the geographical advantage of market assess to the rapid economic growth zones in Asia and South East Asia regions. 5. Labor wages in Malaysia can be either higher or lower as compared to other cocoa producing countries. As Malaysia is progressing to become a developed nation by 2020, labor wages are expected to increase accordingly. However, the anticipated higher wages of labor can be alleviated or compensated by the increase in productivity and efficiency in labor management. 6. Malaysia and other major cocoa producing countries have a fair share of pest and disease problems. These include the CPB in Malaysia and Indonesia, cocoa swollen shoot virus and a serious form of phytophthora disease in West Africa and witches’ broom and monilia disease in Latin America. Technology is available for effective management of the CPB and other pest and diseases in Malaysia and the labor and cost incurred for pest and disease management can be reduced to the minimum through appropriate application of technology and sound management.

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The above analyses indicated that Malaysia has the competitive edge in many aspects. However, in order to remain competitive, Malaysian cocoa planting industry will have to be technology driven with the support of sound management to attain high productivity, efficiency and quality as indicated in the above analysis. Technology development and its utilization through R & D and effective transfer of technology or extension services and other support will continue to play an even more important role in strengthening the foundation of the cocoa planting industry. The competitiveness of Malaysian cocoa planting industry will largely depend on the level of productivity, efficiency and quality that can be attained as called for under the national policy of “Vision 2020”.

Comparative to Other Commodity Crops - The competitiveness of cocoa planting industry as compared to other commodity crops in Malaysia can be considered as follows:

1. The large variation in cocoa prices with a ratio of highest to lowest prices at 5 to 7, which is considered the highest among the commodity crops, provides an excellent opportunity for profit taking in investment. 2. Harvesting of cocoa is a relatively light job and all age groups and sexes can participate in harvesting to earn extra income for the family. Harvesting can be carried out at any time and at different stages of pod ripeness without affecting the quality of cocoa beans. Only 25% of the harvested fresh weight as wet cocoa beans is transported out of the cocoa field. 3. Dried cocoa beans can be stored for a long period and there is no need of special and expensive installation for the storage of cocoa beans. 4 There is always a ready market for cocoa beans at any time. 5. Cocoa has more pest and disease problems than other commodity crops in Malaysia. However, the cocoa producing countries in other regions of the world also face with other serious pest and diseases. More pest and diseases is a threat to the cocoa planting industry world-wide and on the other hand, it can be considered as an opportunity and advantage for those cocoa producing countries who have the technology and management skill for effective and efficient management of the pest and diseases. In this respect, Malaysia has the advantage. 6. Relatively more labor is required in cocoa cultivation mainly due to the need for CPB management. However, the difference in labor requirement as compared to the requirement for the cultivation of other commodity crops is not as wide as often quoted. Furthermore, the labor usage in cocoa cultivation can be more efficiently managed through a good understanding of crop requirement and appropriate application of labor saving technology with sound management. 7. Although “more care and attention” is needed in cocoa cultivation, the effort of “care and attention” provided to the crop is often compensated by a higher productivity and efficiency in return and thus a higher economic return to the growers as incentive.

CONCLUSION

The analyses of the various economic and related factors have come out with the respective indicators for assessment of the perspective for cocoa cultivation in Malaysia. It was indicated that the average price of cocoa is considered very attractive to investment and the large variation in prices creates good opportunity for profit taking. Cocoa pod borer can be kept under control and the labor requirement in cocoa cultivation can be more efficiently managed. There is excellent scope for productivity improvement from the national average of about 0.9 t/ha to 2 t/ha or higher and sustainable high productivity and efficiency can be attained by appropriate utilization of technology through sound management with “commitment and care” or accountability in place.

The prices of cocoa beans and productivity play a significant role in the eventual net farm return and RROI. With a productivity of at least 1.5 t/ha, there is always profit to make. Higher profit margin of up to several hundred percent or higher in RROI is within reach at an attainable productivity of 2 to 3 t/ha and a price range of cocoa beans at between RM4,000/t and RM8,000/t. With a farm size of up to 8 ha per

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family holding and a higher farm productivity, the cocoa smallholders can enjoy good income. Cocoa cultivation in Malaysia has the competitive edge in the international and domestic scenario and the competitive advantage will have to be driven by technology development and utilization with sound management.

The economic indicators presented have conveyed a clear message that cocoa cultivation is very much an economically viable investment. The cocoa planting industry can play a more significant role in contributing to the national economic development under the crop diversification program of the National Agricultural Policy and the Industrial Master Plan II to achieve the objectives of a balanced growth between commodity crops and between the activities of upstream and downstream cocoa industry, and reduction in import of cocoa beans. Furthermore, the requirement of “commitment and care” or personal touch, which can be incorporated in the management system with trace-ability, responsibility and accountability to achieve high productivity, efficiency and even quality in cocoa cultivation, is in-line with the objectives of the national policy of “Vision 2020” which calls for high productivity, efficiency and quality as the pre-requisites for Malaysia to be a developed nation by 2020.

ACKNOWLEDGEMENT

The authors wish to thank Mr. Albert Ling, biometrician of the Malaysian Cocoa Board for the preparation of regression figures.

REFERENCES

Azhar, I. 1995. An overview on the management of key insect pests of cocoa with major emphasis on the cocoa pod borer, Conopomorpha cramerella. The Planters 71: 469-480 Azhar, I., Alias A. and Meriam M. Y. 2000. Research on the management of cocoa pod borer in Malaysia. Proc. INCOPED 3rd International Seminar on Cocoa Pests and Diseases, 16-17 October, 2000. Kota Kinabalu: Malaysian Cocoa Board (MCB) and International Permanent Working Group for Cocoa Pests and Diseases (INCOPED). Chong, G. F., Lam, K. S. and Yap, T. N. (1990). Advances in mechanization of cocoa planting. Proceedings of the MCGC–Malaysian Cocoa Board Workshop on Cocoa Agricultural Research, Pp. 187–196. Malaysian Cocoa Growers’ Council, Kuala Lumpur. Chong, G. F. and Tan, M. W. (1994). Evaluation of tractor mounted/drawn mistblowers and knapsack mistblowers for the application of insecticides in clonal cocoa. Proceedings of International Cocoa Conference: Challenges in the 90’s. 1991. E. B. Tay, M. T. Lee, T. N. Yap, B. I. Zulkarnain, F. T. Thong, S. L. Bong and S. K. Tee (eds.), Pp. 305–313. Malaysian Cocoa Board, Kuala Lumpur. Corley, R. H. V. (1967). Yield potential of plantation crops. Better Crop Intl. 2(2): 10-12. Hidayatullah, H. H. and Hii, C. L. (2000). Assessment of mechanical pruning in cocoa cultivation. Proceedings of Malaysian International Cocoa Conference 1998. M. T. Lee, K. Lamin, L. Johnsiul, D. B. Furtek and F. L. Ening (eds.), Pp. 224–226. Malaysian Cocoa Board, Kuala Lumpur. Ho, C. T., Jamaludin, S. and Wan Ibrahim, A. (1990). Helopeltis research and management in cocoa. In Proceedings of the MCGC – Malaysian Cocoa Board Workshop on Cocoa Agricultural Research, Pp. 120– 130. Malaysian Cocoa Growers’ Council, Kuala Lumpur. ICCO (1992). Quarterly Bulletin of Cocoa Statistics, Vol. XVIII, No. 4, September 1992. International Cocoa Organisation. ICCO (1997). Quarterly Bulletin of Cocoa Statistics, Vol. XXIV, No. 4, December 1997. International Cocoa Organisation. ICCO (2000). Quarterly Bulletin of Cocoa Statistics, Vol. XXVII, No. 1, Cocoa year 2000/2001. International Cocoa Organisation.

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ICCO (2003). Review of possible measures to maintain remunerative cocoa prices. Document EX/118/9. International Cocoa Organisation. ICCO (2004). Quarterly Bulletin of Cocoa Statistics, Vol. XXX, No. 2, Cocoa year 2003/2004. International Cocoa Organisation. Lass, R. A. (1985). Labor usage. In “Cocoa”, 4th edn. G. A. R. Wood and R. A. Lass, Pp. 234–264. Longman, London and New York. Lee, M. T. and Azhar, I. (2004). Accountable management system for high productivity and efficiency in cocoa cultivation : The concept and application. Malaysian Cocoa Board. Lee, M. T. and Chong, T. C. (1987). High yielding cocoa plots – A case study. SASS seminar on Palm Kernel Utilization and Recent Advances in Cocoa Cultivation. 11–13 June, 1987. Lim, K. H., Chung, G. F. and Lam, K. S. (1987). Mechanization in cocoa cultivation. Preprint. Seminar on progress and problems in the cultivation in Negeri Sembilan, Negeri Sembilan. Department of Agriculture. 23 p. MCB. (1991). The cocoa industry in Malaysia – in brief. A booklet published in conjunction with the 1991 International Cocoa Conference organized by the Malaysian Cocoa Board, the Incorporated Society of Planters and Malaysian Cocoa Growers’ Council. Malaysian Cocoa Board, Kuala Lumpur. MCB. (1992). Malaysian cocoa monitor. Vol. 1, No. 1, June 1992. Malaysian Cocoa Board. MCB. (2001). Progress report on Malaysian smallholder cocoa development program 2000. Malaysian Cocoa Board, 2001. MCB. (2000). Malaysian cocoa monitor. Vol. 9, No. 2, December 2000. Malaysian Cocoa Board. MCB. (2004). Annual report 2003. Malaysian Cocoa Board. MCB, (2004a). Malaysian cocoa monitor. Vol. 13, No. 1, June 2004. Malaysian Cocoa Board. Mohd Yusof A. S., Lamin, K., Lee, M. T. and Rosman, R. (2000). High yielding cocoa plots in Peninsular Malaysia – A case study. Proceedings of the Malaysian International Cocoa Conference, 1998. M. T. Lee, K. Lamin, L. Johnsiul, D. B. Furtek and F. L. Ening (eds.), Pp. 45 – 48. Malaysian Cocoa Board, Kuala Lumpur. Taylor, J. G., Ho, C. T. and Lim, T. M. (1982). A mechanized thermal fogging system for controlling Helopeltis theobromae Mill in cocoa. Proceedings International Conference of Plant Protection in the Tropics. K. L. Heong, B. S. Lee, T. M. Lim, C. H. Teoh and Y. Ibrahim (eds.), Pp. 695–702. Malaysian Plant Protection Society, Kuala Lumpur.

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Malaysian Cocoa Journal 24

EXTRACTION OF DNA FROM COCOA (THEOBROMA CACAO L.)

Lea J. Cocoa Biotechnology Research Center, Malaysian Cocoa Board, Lot 4 - 6, Block B Sri Kemajuan Industrial Estate, Mile 6 ½, Tuaran Road, 88400 Kota Kinabalu, Sabah.

Malaysian Cocoa J. 1: 19-23 (2004) ABSTRACT A method was developed for the isolation of cocoa genomic DNA of good quality suitable for use in PCR-based molecular techniques. DNA was isolated from nuclei of young cocoa leaves tissue. Leaf nuclei were isolated by grinding leaf tissue in liquid nitrogen, suspending the powder in buffer and filtering through several layers of gauze cloth. The DNA extracted can be fully digested with restriction enzymes and has been successfully used in the generation of molecular markers using AFLP and SSR techniques.

Key words : DNA, Theobroma cacao, AFLP, SSR

INTRODUCTION

Regardless of the population or DNA marker technique one plans to use, DNA must first be isolated from plants. Several methods of DNA extraction suitable for DNA marker analyses have been developed (Dellaporta et al., 1983, Murray and Thompson 1980; Couch and Fritz, 1990, Lanaud et al., 1995). With each method, the objective is: develop a simple, rapid procedure that yields DNA of good quality and quantity from small amounts of starting material. Simplicity and speed are essential for processing large numbers of plants. Small amount of starting material is most important when larger quantities are hard to obtain.

Unlike easily extracted plants such as rubber (Hevea brasiliensis) and Arabidopsis (Arabidopsis thaliana), cocoa tissues contain high levels of mucilage, tannins, and other polyphenolic compounds. When cells are disrupted, these cytoplasmic compounds come into contact with DNA, RNA and all other cellular components (Loomis, 1974). In their oxidized forms, polyphenols covalently bind to DNA giving it a brown colour and making it useless for most research applications.

Several published extraction methods have been tried and found unsuitable for cocoa, due to the high levels of gums (charged polysaccharides) and polyphenolic compounds within the tissues. Other published protocols developed specifically for cocoa or plants with high amounts of polyphenolic compounds are complex, require extensive centrifugation steps, often with the use of expensive cesium chloride gradients and expensive equipment (Couch and Fritz, 1990; Figuiera et al., 1992, 1993; Lanaud et al., 1995). After trying several modified protocols, one DNA extraction method was developed for cocoa molecular marker research.

In this study, I attempted to develop a simple genomic DNA extraction technique (especially for cocoa) that produces unsheared, good quality DNA without the use of expensive laboratory equipment and chemical.

MATERIALS AND METHODS

Plant Materials - Plant materials used were progenies (30 trees) of clone Parinari 7 (PA 7) crossed with Nanay 33 (NA 33). The plant materials used in this research were obtained from Quoin Hill Agriculture Station, Tawau, Sabah.

Collection and Preparation of Plant Materials - The plant material used in the study was fully expanded, young cocoa leaves. Too young leaves have a high content of mucilage whereas too old leaves have a high

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level of polyphenolic compounds. Most leaves had to be shipped overnight to the laboratory. After leaves were harvested, they were cleaned, sandwiched between moist newspapers in plastic bags for shipping. Upon arrival, leaves were rinsed several times with tap water and finally with MiliQ water (Millipore pure water) before being pat-dried with paper towels. Midribs were removed before being frozen with liquid nitrogen. The frozen leaves were then freeze-dried for several hours until the leaves were fully dry. The freeze-dried leaves were sealed in plastic bags for storage at -70oC.

DNA Extraction Protocol - Half gram of freeze-dried leaf sample was weighed and immersed in liquid nitrogen. Leaf tissue was then ground to a fine powder under liquid nitrogen with mortar and pestle. Then 75 ml of cold NEB buffer [300 mM sucrose, 5 mM MgCl2 (Sigma, USA), 50 mM TrisCl (Sigma, USA) pH 8.0, 1% (v/v) Triton X-100 (Sigma, USA), 2% polyvinylpyrrolidone 40 (PVP40) (Sigma, USA), 0.5% (w/v) sodium metabisulphite (Sigma, USA)] was added to the leaf powder. As the NEB buffer and leaf tissue began to thaw, they were ground to a gelatinous homogenate. When fully thawed, the homogenate was filtered through a 4-layer sterilized gauze cloth and left to stand in ice for 20 minutes. The filtrate was then centrifuged (Heraeus Sepatech, Germany) at 7900RCF for 25minutes at 4oC. The supernatant was discarded and tubes were drained inverted on paper towels to remove as much supernatant possible. The pellet was resuspended in 25 ml NEB buffer and centrifuge as above a further two times. After the final NEB washing, supernatant was discarded and the DNA was extracted from the nuclear pellet by addition of 1.8 ml RBS buffer (30 mM TrisHCl pH8.0, 10 mM EDTA, 1% (v/v) Sarkosyl, 2% (w/v) PVP40, 0.5% (w/v) sodium metabisulphite). The nuclear pellet was resuspended in RBS buffer and 10 µL of Proteinase K (Sigma, USA) in RBS buffer (5 mg/ml) was added, followed by incubation for 3 hours at 37oC or 1 hour at 56oC with occasional mixing by inversion. The mixture was treated with one time equal volume of chloroform/isoamyl alcohol (24:1, v/v) followed by one time equal volume of phenol/chloroform/isoamyl alcohol (25:24:1, v/v), then a final extraction with equal volume of chloroform/isoamyl alcohol (24:1, v/v). Two volumes of cold 100% ethanol was added to the final aqueous phase then incubated for 1 hour at - 20oC. DNA was recovered by centrifugation (11,000 RCF, 10minutes, 4oC), supernatant removed and stand tube in an inverted position on a paper towel to allow all of the fluid to drain away. DNA pellets were washed twice in 70% (v/v) ethanol and dried under vacuum. Pellets were then dissolved in 100 µl TE buffer (10 mM Tris.Cl, pH 8.0 and 1 mM EDTA, pH 8.0). Residual RNA was removed by incubation with 0.2 mg/ml RNAse A (Sigma, USA) (final concentration) at 37oC for 1 hour.

DNA solutions were then checked for quality and quantity. Three micro liters of DNAs (150-300 ng total) were cleaved with 1 unit of EcoRI (NEB, USA) at 37oC for 1 hour, electrophoresed in 0.75% agarose gel in 1X TBE buffer (89 mM Tris base, 89 mM boric acid, 2 mM EDTA). DNA suspensions were also quantified using the Hoechst 33258 dye-binding assay (Bio-Rad, USA) on a DNA Fluorometer TKO100 (Hoefer Scientific Instruments, USA) according to manufacturer’s instruction.

Quality Checking and Quantity Estimation Using Agarose Gel Electrophoresis - DNAs were first checked for quality by agarose gel electrophoresis. Three (3) µl of DNA suspension and 2 µl 6X loading solution (0.25% (w/v) bromophenol blue and 15% (w/v) Ficoll 400) was mixed for loading on 0.7% agarose gel. Another set of DNAs were digested with EcoRI for at least 1 hour at 37oC. Undigested and cleaved DNAs were run side by side. The λHindIII molecular ladder (Bio-Rad, USA) was also included in the gel. The DNA quality was checked by looking: a. uncut DNA band is clear, more than 23kbp with minimal shear DNA (the smear part below the band), and with minimal DNA stuck in the well. b. Cleaved DNA has a smooth smear and sometimes with faint bands.

The DNA quantity was estimated by comparing with the brightness of the known amounts of DNA in the molecular weight marker bands.

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Quantitation of DNA Using the DNA-binding Fluorochrome Hoechst 33258

A Hoefer DNA Fluorometer Model TKO100 (Hoefer Scientific Instruments, USA) was used to quantify DNA using the bis-benzamine or commonly known as Hoechst 33258 assay (DNA Quantitation Kit, Bio-Rad USA) according to the manufacturer’s instruction. The assay has an advantage of minimizing the interference of protein, RNA, and nucleotides (compared to common spectrophotometry). A standard curve were drawn using the TableCurve 2D (SPSS Sciences, USA) computer program to estimate the concentration of DNA.

Random Amplified Polymorphic DNA (RAPD) - Several progenies from the crossed population were screened with three random primers (Advanced Biotechnologies, UK). Polymerase chain reaction (PCR) contained 20 ng DNA, 0.13 mM dNTP (Gibco-BRL, USA), 5 M random primer (Advanced Technologies, UK), 1.5U Taq polymerase (Gibco-BRL, USA ), 1X PCR buffer [20 mM Tris-HCl pH8.4, 50 mM KCl, 2.5 mM MgCl2, 0.13% (v/v)Tween20, 0.13% (v/v) Nonidet P40], made to a total volume of 20 l with sterile distilled water. Two drops of mineral oil were added to the tubes to prevent evaporation of PCR reactions.

Amplification was performed in a Perkin Elmer Cetus DNA Thermocycler model 2400 (Perkin Elmer, USA) for 25 cycles of 0.5 min at 94oC, 1 min at 36oC, and 2 min at 72oC and a further 5 min extension time after the 25 cycles at 72oC. RAPD fragments were separated on a 2 % (w/v) agarose 1x TBE gel.

RESULTS

The amount of DNA obtained per gram of freeze-dried cocoa leaf varied from 8 ug to 20 ug. The DNA is clear or white, i.e. there is no visible coloration although some yellow tint was observed if the leaf tissues have been kept for several days at ambient temperature prior to freeze-drying (example when the leaf was sent through courier service). The DNA is mostly of high molecular weight, more than 23kbp. Isolated cocoa DNA is fully digestable with EcoRI and shows no visible RNA contamination as determined by agarose gel electrophoresis. There is either no or very little mucilage co-purified with the DNA as seen by DNA stuck in the wells, and the amount of DNA stuck with the mucilage seems to decrease after digestion with restriction enzyme (Figure 1). The best stage of leaf development for DNA extraction was when it had fully expanded but still soft. Too young leaves contained a high content of mucilage whereas too old leaves contained high level of polyphenolic compounds. Complete resuspension of the nuclear pellet in buffer before centrifugation was essential to ensure a high yield of DNA.

A B C D

Figure 1. Gel electrophoresis of extracted DNA using different procedures. For each pair, the left side is undigested DNA and the right side is DNA digested with EcoRI A= the current DNA extraction method product, B = using CTAB method, C= using DNeasy Plant Mini Kit and D = according to Dellaporta, Wood and Hicks, 1983.

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RAPD Analyses

RAPD markers were obtained using the DNA extraction method outlined above. The bands can be distinguished clearly from each other and are easy to score (Figure 2).

Figure 2. Gel electrophoresis of RAPD products. The same primer pair was used for the first three progenies (from left) and another primer pair was used for the other two progenies (on the right). The right side is the λHindIII molecular ladder.

DISCUSSION

In an attempt to isolate good quality cocoa DNA, several published protocols for isolation of nuclear DNA were tried. The protocols tried mostly produced DNA that was brown colored due to polyphenol contaminations or DNA that was highly contaminated with mucilage (Couch and Fritz, 1990). Moreover most of the protocols use expensive or specialized equipment such an ultracentrifuge which may not be available in small budget laboratories and expensive chemicals such as cesium chloride and CTAB (cetyltrimethylammonium bromide).

Consequently, a simple DNA extraction protocol was developed that yielded a reasonable amount of DNA which was polyphenol and mucilage free. The DNA is of good quality and can be used as a template for PCR-based molecular marker techniques. Because different plants can vary considerably in the amount and number of secondary metabolites they produce, it is unlikely that any one technique for DNA extraction can be developed (Loomis, 1974). It is likely, however, that this developed DNA extraction protocol can be used to isolate nuclear DNA from a variety of other plant species especially high in polyphenols, tannins and mucilage.

CONCLUSION

Good quality cocoa DNA has been isolated from nuclei of cocoa leaves. The DNA prepared was larger than 23kbp in size and was clean, clear in colour, showed no visible RNA contamination, and no or very little mucilage co-purified with it. The DNA was also fully digestable with EcoRI and suitable for use in generating PCR-based molecular markers.

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ACKNOWLEDGEMENT

I would like to thank the Director General of MCB for permission to published this paper. I would also like to thank MCB breeders who supplied the plant materials and Ms Sairan Asim who assisted in the laboratory. Thanks also to the anonymous reviewer for critical reviewing of manuscripts. This work was supported by a grant from the Malaysian Government’s Intensified research in Priority Areas (IRPA) program (IRPA no. 01-04-07-0302).

REFERENCES

Couch, J. A., Fritz, P. J. (1990). Isolation of DNA from plants high in phenolics. Plant Molecular Biology Reporter 8: 8-12. Dellaporta, S. J., Wood, J., Hicks, J. B. (1983). A plant DNA mini preparation: version II. Plant Biology Reporter 1: 19-21. Figuiera, A., Janick, J., Goldsbrough, P.(1992). Genome size and DNA polymorphism in Theobroma cacao. J. Am. Soc. Hortic. Sci. 17: 673-677. Lanaud, C., Risterucci, A. M., N’Goran, J. A. K., Clemet, D., Flament, M. H., Laurent, V., Falque, M. (1995). A genetic linkage map of Theobroma cacao L. Theor. Appl. Genet 91: 987-993. Loomis, W. D. (1974) Overcoming problems of phenolic and quinines in isolation of plant enzymes and organelles. Method in Enzymology 31(A): 528-544. Murray, M. G., Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acid Res 8: 4321-4325.

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COCOA GENETIC LINKAGE MAP CONSTRUCTION TOWARDS QTL MAPPING

Lea J.1, K. Lamin2 and D. B. Furtek1 1 Cocoa Biotechnology Research Center, Malaysian Cocoa Board, Lot 4 - 6, Block B Sri Kemajuan Industrial Estate, Mile 6 ½, Tuaran Road, 88400 Kota Kinabalu, Sabah. 2 Malaysian Cocoa Board, Locked Bag 211, 88999 Kota Kinabalu, Sabah, Malaysia.

Malaysian Cocoa J. 1: 24-27 (2004) ABSTRACT In order to assist breeders with selecting cocoa plants with superior adult traits - such as yield, resistance to diseases, and flavor potential – while plants are still at the seedling stage, a genetic linkage map is being constructed using 100 siblings from a cross between UIT2 and NA33 using amplified fragment length polymorphic (AFLP) DNA markers. The sibling population is more than 10 years old and has already been scored for yield, resistance to Phytophthora pod rot (black pod), vascular streak dieback (VSD) disease, precocity, and bean size. AFLP markers labeled with fluorescent dyes are being generated and separated according to sizes using an automated sequencing and DNA analysis machine, MegaBACE 500 (Amersham Biosciences, USA). To date, four AFLP primer-pairs have been used to screen the mapping population. A total of 45 segregating markers have been identified. A majority of the markers were found to be inherited in a Mendelian manner, indicating their suitability for use in the construction of a linkage map. A framework map was generated using these markers.

Key words: Cocoa, AFLP, Genetic Linkage Map, QTL, JoinMap, Thebroma cacao

INTRODUCTION

SelS ecting cocoa clones for breeding or breeding for trees with superior adult traits can be a difficult task for breeders and requires much, perhaps ten years or more. In addition, much land, and money are required. The availability of a cocoa genetic linkage map with desirable traits like high yield, disease resistant, high butter content, etc. will enable breeders to identify superior plants by just looking at the DNA markers linked to the desirable traits even at the seedlings stage. Seedlings found to have the desirable traits can be planted in the field whereas those do not can be discarded. This will greatly help in saving labor costs, land-space, and time. Breeders will not have to wait many years assessing the plants (and discard the great majority that do not have desirable traits). A cocoa linkage map developed with QTL (quantitative trait loci) for valuable traits and molecular markers will help cocoa breeders to identify superior plants by just looking for the DNA markers linked to the loci for desired trait. In assessing cocoa seedlings, breeders will just have to collect young cocoa leaves and send them to the laboratory. At the laboratory, researchers extract DNA, then generate molecular markers using primer pairs known to produce the markers closely linked to the desired traits. Results can be made available to breeders within one or two weeks. In the present study, we concentrated on a preliminary genetic linkage map of cocoa using AFLP markers.

MATERIALS AND METHODS

Plant Materials - A segregating population of 100 individuals from a cross between UIT2 and NA 33 was used. The progeny are still standing in Tawau, Sabah, Malaysia.

DNA Extraction - Half gram of freeze-dried leaf samples were weighed and ground in liquid nitrogen. Leaf powder was added to cold NEB buffer [300 mM Sucrose, 5 mM MgCl2 (Sigma, USA), 50 mM TrisCl (Sigma, USA) pH 8.0, 1% (v/v) Triton X-100 (Sigma, USA), 2% Polyvinylpyrrolidone 40 (PVP40) (Sigma, USA), 0.5% (w/v) sodium metabisulphite (Sigma, USA)], mixed into a gelatinous homogenate, and left to thaw. After the homogenate had fully thawed, it was filtered and left to stand in ice for 20 minutes. The filtrate were then centrifuged (Heraeus Sepatech, Germany) at 7900RCF 3,000 rpm for 25 minutes at 4oC. The supernatant was discarded and the pellet resuspended in 25 ml NEB buffer and

Malaysian Cocoa Journal 30

centrifuged as above a further two times. After the final centrifugation, the supernatant was discarded and the DNA was extracted by resuspending the nuclear pellet in 1.8 ml RBS buffer [30 mM TrisHCl pH8.0, 10 mM EDTA, 1% (v/v) sarkosyl, 2% (w/v) PVP40, 0.5% (w/v) sodium metabisulphite] then adding 10 µl of Proteinase K (Sigma, USA) in RBS buffer (5 mg/ml). The mix was incubated for 3 hours at 37oC or 1 hour at 56oC with occasional mixing by inversion. This was followed by a phenol:chloroform:isoamyl extraction. The DNA was then precipitated with 2X volume 100% ice-cold ethanol followed by washing with 70% cold ethanol before being vacuum dried. Pellets were then resuspended in 100 µl TE buffer (10 mM TrisCl, pH 8.0 and 1 mM EDTA, pH 8.0). RNA was removed by incubation with a final concentration of 0.2 mg/ml RNAse A (Sigma, USA) at 37oC for 1 hour and kept for at least 3 days in 4oC before storing indefinitely at -20oC.

AFLP Analysis - There are four steps in this analysis: 1) restriction enzyme digestion and ligation, 2) preamplification, 3) selective amplification, and 4) electrophoresis in an Amersham MegaBACE 500 DNA sequencing and fragment analysis machine (hereafter referred to as MegaBACE).

Restriction enzyme digestion and ligation was done simultaneously in one tube (1X T4 DNA ligase buffer + ATP, 0.05 M NaCl, 0.05mg/ml BSA, 1 µl EcoRI adaptor, 1 µl MseI adaptor, 0.2 µ/µl MseI enzyme, 0.5 µ/µl EcoRI enzyme, 4 µ/µl T4 DNA ligase and 250 ng genomic DNA) incubated at 37oC for 2 hours before diluted 20X with 0.1X TE buffer.

Preamplification was done by combining in a tube 1 X (NH4)2SO4 PCR buffer, 2 mM MgCl2, 0.2 mM dNTP mix, 1.0 ul preamplification primers (Applied biosystems), 0.05 µ/µl Taq DNA polymerase and 4ul of the diluted restricted and ligated DNA for a final volume of 20ul. PCR was performed according to the temperatures cycles as described in the Applied Biosystems Plant AFLP Manual. PCR product was then diluted 10X with 0.1X TE buffer

Selective amplification was done in PCR reaction mixture of 1 X (NH4)2SO4 PCR buffer, 2 mM MgCl2, 0.2 mM dNTP mix, 1.0 µl EcoRI primer, 1.0 µL MseI primer, 0.05 µ/µl Taq DNA polymerase and 3.0 µl diluted preselective amplified DNA. PCR was performed for 30 cycles as described in the Applied Biosystems Plant AFLP Manual.

Two (2.0) µl of selective amplified DNA was mixed with 1.0 µl molecular weight standard (ET550-ROX), and 17.0 µl water before run into an Amersham MegaBACE 500 DNA sequencing and fragment analysis machine.

Map Construction - AFLP loci were scored as dominant markers i.e. segregation was scored on the basis of presence or absence of markers.

The markers were arranged according to their sizes within a 1 bp range in MsExcelTM. Polymorhic fragments that were present in one or both parents were recorded and their segregation patterns among progenies were tested for Mendelian segregation using a Chi Square (χ2) test. In this population, the predicted segregation pattern should be either 1:1 or 3:1. This is because AFLP markers are scored as dominant markers i.e. as either absent or present.

Table 1 illustrates the types of Mendelian segregation of AFLP markers expected in the mapping population. Linkage map construction was carried out using JOINMAPTM Version 3.0 (Van Ooijen and Voorrips, 2001). The population was analysed as a population resulting from a cross between two heterozygous diploid parents. The population type used in the analyses were type code CP as indicated in the JOINMAPTM manual (Van Ooijen and Voorrips, 2001). Linkage was identified by stepwise lowering of the LOD score from ten to two with a maximum recombination frequency of 0.450. LOD score of 4.0 was the lowest stringency at which acceptable linkages were performed. Map distances were calculated using the Kosambi function (Kosambi, 1944).

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Table 1: Segregation of AFLP markers expected in the mapping population

Parental Genotypes Expected Mendelian ratio

Aa x aa 1:1 (female) Aa x Aa 1:1 (male) Aa x Aa 3:1

RESULTS AND DISCUSSION

The mapping population so far has been screened using four AFLP primer pairs (TA-CTT, TG- CTA, TC-CAG and AG-CAG). A total of 45 loci were scored and found to be informative and following Mendelian segregation patterns. Further generation of markers is in progress.

A total of 38 markers were used to construct the map at a minimum LOD score of 4.0. Thirty eight (38) markers were assigned to 23 groups. However only 5 linkage groups were able to be constructed (group 1, 11, 16, 18 and 19) with a total of 17 loci (Figure 1). The remaining markers were unlinked to any other marker at LOD 4.0. Ultimately, the map contain 10 linkage groups, corresponding to the 10 pairs of chromosomes (Lanaud et al., 1995). AFLP analysis using different primer pairs are currently being used to generate more markers.

1 11 16 18 19

0 MCBaflp2/10 0 MCBaflp5/12 0 MCBaflp4/1 0 MCBaflp4/6 0 MCBaflp10/1

5 MCBaflp2/14

9 MCBaflp2/7

15 MCBaflp2/21 15 MCBaflp5/2 16 MCBaflp2/18

20 MCBaflp10/8 21 MCBaflp4/2

25 MCBaflp2/20

29 MCBaflp5/10 30 MCBaflp4/12 32 MCBaflp2/13

44 MCBaflp5/7

Figure 1: Our first preliminary cocoa genetic linkage map

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ACKNOWLEDGEMENT

We thank the Director General of MCB for permission to published this paper. We also thank the MCB breeders (Mr. Francis Aloysius and Mr. Muhammad Jaafar H.) who supplied the plant materials and Mrs. Sairan Asim for help in the laboratory. This work was supported by a grant from the Malaysian Government’s Intensified Research in Priority Areas (IRPA) program (IRPA no. 01-04-07-0305).

REFERENCES

Alias, A.A., Mohd Dahalan, A., Kasran, R. and Furtek, D.B. (2003). Molecular Fingerprinting of Cocoa Clones From MCB Hilir Perak. Proceeding 8th Malaysian Cocoa Board Senior Staff Conference. 23- 25 September 2003.pp. 45-48. Kosambi, D. D. (1944) The Estimation of Map Distance From Recombination Values. Ann. Eugen 12:172- 175. Lanaud, C., Risterucci, A. M., N’Goran, J. A. K., Clement, D., Flament, M. H., Laurent V., Falque M. (1995) A Genetic Linkage Map of Theobroma cacao L. Theor. Appl. Genet. 91:987-993. Van Ooijen, J. W and R. E. Voorrips, (2001), JoinMap® 3.0, Software for calculation of genetic linkage maps. Plant Research International, Wageningen, The Netherlands.

Malaysian Cocoa Journal 33

RECURRENT EMBRYOGENESIS AND IMPLICATIONS FOR GENE TRANSFER IN THEOBROMA CACAO L.

Tan C. L. and D. B. Furtek Cocoa Biotechnology Research Center, Malaysian Cocoa Board, Lot 4 - 6, Block B Sri Kemajuan Industrial Estate, Mile 6 ½, Tuaran Road, 88400 Kota Kinabalu, Sabah.

Malaysian Cocoa J. 1: 28-35 (2004) ABSTRACT In order to establish a recurrent somatic embryogenesis system for Theobroma cacao L., several factors were evaluated. The influence of genotype was significant among the cocoa clones studied. Among the growth regulators studied, 0.1 mgl-1 N6-(2-isopentenyl) adenine (2iP) treatment produced the highest percentage of secondary somatic embryos. The percentage of tertiary embryogenesis also did not differ significantly from secondary embryogenesis in this study. Microscopic examination revealed that secondary embryos proliferate from epidermal tissue of the primary embryo.

Key words: Theobroma cacao L, Recurrent embryogenesis, Secondary somatic embryogenesis, Cocoa.

Abbreviations: 2,4-D: 2,4-dichlorophenoxyacetic acid, NAA: 1-Naphthaleneacetic acid, SEM: Scanning electron microscope, 2iP: N6-(2-isopentenyl)adenine, KIN: Kinetin, TDZ: Thidiazuron, BAP: 6- Benzylaminopurine

INTRODUCTION

Somatic embryogenesis is the development from somatic cells, through an orderly series of characteristic morphological stages, of structures that resembles zygotic embryos. Recurrent somatic embryogenesis, which comprises successive cycles of somatic embryogenesis induced on explants of regenerated plants, has been successfully used for the transformation of soybean using microprojectile bombardment and of alfalfa using inoculation with Agrobacterium (Vasic et al., 2001). Secondary somatic embryogenesis is a developmental pathway whereby new somatic embryos are initiated from somatic embryos. As an experimental system, it has certain advantages when compared to primary somatic embryogenesis – such as high multiplication rate, independence of an explant source, and repeatability. Furthermore, embryogenicity can be maintained for prolonged periods of time by repeated cycles of secondary embryogenesis, as in recurrent embryogenesis. Regeneration characteristics such as epidermal and single-cell origin of somatic embryos favour the use of secondary somatic embryogenesis for plant transformation (Martenelli et al., 2001).

Several investigators (Alemano et al., 1996, Sondahl et al., 1993) have observed that secondary embryogenesis occurred spontaneously during the process of somatic embryogenesis of cocoa from immature flower explants. This paper attempts to evaluate factors (genotype, growth regulators, carbon source) affecting secondary embryogenesis and subsequently establishing a recurrent embryogenesis system for cocoa. Microscopic examination was also carried out to reveal the cellular events occurring during secondary embryogenesis of cocoa.

MATERIALS AND METHODS

Initial Plant Materials - Plant materials were obtained from bud-grafted field grown cocoa trees of clones KKM 9, PBC 137, QH 1560 and KKM 19 planted at the Center for Cocoa Biotechnology Research, Malaysian Cocoa Board, Kota Kinabalu, Sabah, Malaysia.

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Culture Conditions

Primary Somatic Embryogenesis - Unopened flower buds (4 – 6 mm in length) were surface sterilised by washing with tap water, immersion in 5% (v/v) “Clorox®” bleach (Lever Brothers, Malaysia) for one minute and rinsing in sterile reverse osmosis water three times. Staminodes were extracted, using scalpels and forceps, following removal of sepals and petals. For callus initiation, staminodes were cultured in 9 cm plastic Petri dishes (Mutiara Medical Ltd., Malaysia; 20/dish) with each dish containing 30 ml of callus initiation (MIM42n) medium. Dishes were sealed with Parafilm® (Pechiney Plastic Packaging., Menasha, WI 54952, USA) and incubated for 3 – 4 weeks in the dark at 26oC ± 2oC. Calli were then subcultured onto 30 ml of differentiation MEM22a medium (Tan and Furtek, 2003) for 6 – 8 weeks in the dark with transfer to the same medium after 4 weeks. MIM42n medium is composed of Driver and Kuniyuki (DKW) basal salts (Duchefa, Biochemie BV, The Netherlands) 20 gl-1 glucose, Murashige and Skoog (MS) vitamins, 2 gl-1 2,4-dichlorophenoacetic acid (2,4-D), 0.1 gl-1 N6-[2-isopentenyl]adenine (2-iP), 1.0 mgl-1 NAA, 500 mgl-1 glutamine and 2 gl-1 Phytagel (Sigma), pH5.5. Globular to cotyledonary stage somatic embryos were removed from the calli and maintained on MEM22a medium with subculture every 2 – 3 weeks.

Secondary Somatic Embryogenesis - Cotyledons of primary somatic embryos (ca. 6-8 weeks from day of initiation) were cut into pieces of about 4 mm2. These cotyledon pieces were transferred onto initiation medium as in primary somatic embryogenesis for about 3 weeks and subsequently to MEM22a medium for secondary embryos differentiation.

Conversion into Plant - Somatic embryos (>10mm in length) with clearly defined shoot and root axes were transferred individually to 50 ml of MGM medium for germination. MGM medium consisted of 1/5 X DKW salts, 5 gl-1 sucrose, 10 gl-1 glucose, 100 mgl-1 myo-inositol, 2 mgl-1 glycine, 2.2 gl-1 Phytagel (Sigma), pH5.8. Media were dispensed onto 250 ml glass jars (Biocraft, Singapore) and covered with Suncaps (Sigma) for ventilation. The cultures were kept in the light (16 h photoperiod, daylight fluorescent tubes, 25 µmol m-2 s-1) at 25oC ± 2oC.

Experimental Procedures and Statistical Analyses - Cotyledon tissues from primary embryos of clones KKM 9, PBC 137 and KKM 19 were cut into 4 mm2 pieces and cultured on MIM42n for 3 weeks. These tissues were subsequently transferred to MEM22a medium for about 6-8 weeks with subculture after every 3 weeks. The experiments on recurrent embryogenesis were aimed at determining the effects of genotype, growth regulators, carbon source and efficiency of secondary versus tertiary embryogenesis. The experiments were arranged in a complete factorial design. After eight weeks, the number of explants producing embryos was recorded. Each treatment consisted of at least five replicate (20 explant/Petri dish). Means and standard errors (S.E.M.) were used throughout and statistical significance among values were assessed using ANOVA (Snedcor and Cochran, 1989) incorporating the post-hoc Tukey-Honestly significantly difference (Tukey HSD) test using a commercial statistical software (Minitab®). A probability of <0.05 was considered significant.

Histological Examination - Histological sections of primary embryos bearing secondary embryos were made from 7-8 week-old embryos on MEM22a medium. Primary embryos with secondary embryos at different stages of development were fixed in FAA (formalin:glacial acetic acid:ethanol, 5:5:90, v:v:v). They were then passed through an ethanol-tertiary butanol series and embedded in wax. Sections of 8-10 µm were stained with safranin and fast green.

Scanning Electron Microscopy - Somatic embryos (3 – 9 weeks after culture initiation) were frozen in liquid nitrogen for 5 minutes and mounted on a scanning electron microscope (SEM) support and kept at – 194oC in a vacuumed chamber. The samples were covered with a thin layer of gold (250-300 A) using a sputter coater and examined with a JEOL 25-S-II SEM at 10 Kv.

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RESULTS AND DISCUSSION Genotype

Clone KKM 9 exhibited the highest percentage of explants producing embryos (35.3%) (Figure 1A). This is followed by KKM 19 (30.0%) and PBC 137 (11.7%). Significant difference (P<0.05) in results was observed among the various clones, indicating an influence of genotype on secondary embryogenesis frequencies.

The average number of somatic embryos per responsive explant was also highest in clone KKM 9 (8.1) (Figure 1B). This is followed by clone KKM 19 (3.0) and PBC 137 (2.9). Significant differences (P<0.05) was observed among the different clones. Secondary embryos were morphologically similar to primary embryos. The influence of genotype on embryogenesis was also observed in many other plant species, such as apple (Höfer 2004), Quercus robur (Toribio et al. 2004) and egg plant (Kantharajah and Golegaonkar 2004). Genotypic effect was also observed in primary embryogenesis in cocoa (Tan and Furtek 2003). The present study indicates that genotypic effects also influence the efficiency of recurrent embryogenesis in cocoa.

40

30

20

10 embryos (%)

Explant producing 0 PBC137 KKM19 KKM9 Cocoa clones

Figure 1A. Effect of different genotypes on the percentage of explants producing secondary embryos in cocoa. Values are means of five experiments and standard error.

c 10 8

of somati 6 4 embryos 2 0

Average no. PBC137 KKM19 KKM9 Cocoa clone

Fig. 1B Effect of genotype on the average number of somatic embryos per responsive explant. Values are means of five experiments and standard error.

Malaysian Cocoa Journal 36

Growth Regulators

Among the treatments, the highest percentage of explants producing somatic embryos was observed from treatment with only cytokinin. Treatment B (0.1 mgl-1 2iP) exhibited the highest percentage of explant, producing somatic embryos (16.7%) (Figure 2A), this is followed by treatment D (0.1mgl-1 KIN) (4.4%). Those treatments with added auxin, whether 2,4-D or NAA, have a lower rate of embryogenesis or no embryogenesis at all. Callus was observed in all the treatment with auxin (NAA or 2,4-D). However, somatic embryos appeared to be induced directly from explants and without a callus stage. Similary, direct somatic embryogenesis also appear to occur from immature zygotic embryos of cocoa via a “budding” process in which cells of the hypocotylary epidermis develop to mimic the normal stages of embryogenesis including the development of a suspensor (Kononowicz et al., 1984).

30 t an l

) of 25

(% 20 e g

ve exp 15 ta si n n

e 10 o c r 5 Pe resp 0 ABCDEFGH I JKL Treatment

Figure 2A. Effect of phytohormones on secondary embryogenesis in clone KKM9. Values are means of five experiments and standard error. A = 0.1 mgl-1 TDZ, B = 0.1 mgl-1 2iP, C = 0.1 mgl-1 BAP, D = 0.1 mgl-1 KIN, E = 2 mgl-1 2,4-D, 0.1 mgl-1 TDZ, F = 2 mgl-1 2,4-D, 0.1 mgl-1 2iP, G = 2 mgl-1 2,4-D, 0.1 mgl-1 BAP, H = 2 mgl-1 2,4-D, 0.1 mgl-1 KIN, I = 2 mgl-1 NAA, 0.1 mgl-1 TDZ, J = 2 mgl-1 NAA, 0.1 mgl-12iP, K = 2 mgl-1 NAA, 0.1 mgl-1 BAP, L = 2 mgl- 1NAA, 0.1 mgl-1KIN. Each treatment was conducted on no less than 20 explants.

The addition of 2iP also resulted in the highest average number of somatic embryos per responsive explant (1.2) (Figure 2B) compared to other treatments. This is followed by treatment with kinetin (Treatment B) (0.7); with treatment with TDZ (Treatment D) being the least (0.2). Generally, there is a large variation among growth regulators used to induce somatic embryogenesis in dicot species. Zygotic embryos and flower associated explants initiated embryos in a greater proportion of species on cytokinin supplemented or growth regulator-free media than vegetative explants. In general, secondary embryogenesis requires no growth regulators in species with cytokinin-driven primary embryogenesis. Whereas, continuous exposure to growth regulators is needed in species with cytokinin/auxin or auxin- driven primary embryogenesis (Raemakers et al., 1995).

Malaysian Cocoa Journal 37 e v

f 2 si o n o er

b 1.5 sp t m e n u r a r l

e 1 e n exp s p ag

yo 0.5 r ver b A

em 0 ABCDEFGH I JKL Treatment

Figure 2B Effect of phytohormones on the average number of embryos per responsive explant in cocoa clone KKM9. Values are means of five experiments and standard error. A = 0.1 mgl-1 TDZ, B = 0.1 mgl-1 2iP, C = 0.1 mgl-1 BAP, D = 0.1 mgl-1 KIN, E = 2 mgl-1 2,4-D, 0.1 mgl-1 TDZ, F = 2 mgl-1 2,4-D, 0.1 mgl-1 2iP, G = 2 mgl-1 2,4-D, 0.1 mgl-1 BAP, H = 2 mgl-1 2,4-D, 0.1 mgl-1 KIN, I = 2 mgl-1 NAA, 0.1 mgl-1 TDZ, J = 2 mgl-1 NAA, 0.1 mgl-1 2iP, K = 2 mgl-1 NAA, 0.1 mgl-1BAP, L = 2 mgl-1NAA, 0.1 mgl-1KIN.

Carbon Source

Fructose and maltose are the only two carbon sources that induced secondary embryogenesis in this study. Both resulted in 1% embryogenesis in clone QH 1560, whereas the other clones (KKM 9 and PBC 137) did not respond (Table 1).

Table 1. Effect of different carbon source in expression media on secondary embryogenesis in of clones KKM 9, QH 1560 and PBC 137. The total number of explants for each treatment was 20 and each treatment were repeated at least three times.

Explant bearing embryos (%) KKM 9 QH 1560 PBC 137 Sucrose 0 0 0 Fructose 0 1.0 ± 1.0 0 Maltose 0 1.0 ± 1.0 0 Mannitol 0 0 0 Galactose 0 0 0 Lactose 0 0 0 Glucose 0 0 0 Values were mean ± standard error.

Secondary Embryogenesis Versus Tertiary Embryogenesis

The efficiency of secondary and tertiary embryogenesis was studied in clone PBC 137. The percentage of explants bearing embryos was slightly higher in tertiary embryogenesis (17.5%) compared to secondary embryogenesis (11.7%). The average number of embryos per explant remains the same in secondary (2.90) and tertiary embryogenesis (2.52) (Table 2). The present cultures have been maintained on media lacking plant growth regulators for more than three years. New somatic embryos formed with

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each subculture to fresh medium starting a recurrent process of embryogenesis. This provides a ready source of somatic embryos for genetic transformation and propagation purposes.

Table 2. Efficiency of secondary and tertiary embryogenesis in clone PBC 137. The total number of explants for each treatment was 20 and each treatment was repeated at least three times.

Secondary Tertiary embryogenesis embryogenesis Percentage of explants bearing embryos 11.7 ± 3.33 17.5 ± 9.06 Average number of embryos per responsive explants 2.90 ± 1.07 2.53 ± 0.92 Values were mean ± standard error.

Figure 3. Secondary embryogenesis in cocoa. (a) Secondary embryogenesis from cotyledon tissue of clone KKM 9 on MEM(22a) medium (bar = 1.2 mm), (b) Secondary embryogenesis from hypocotyl tissue of clone KKM 9 on MEM(22a) medium (bar = 0.7 mm), (c) Six-week old germinated plantlet derived from a secondary somatic embryo of clone KKM 9 on MGM medium (bar = 4.0 mm), (d) Three-month old ex vitro plant derived from secondary and tertiary somatic embryos of clone KKM 9 and PBC 137 (bar = 8.9 mm), (e) SEM examination of a 7-8 week old culture of secondary embryos of clone KKM 9; arrow indicates secondary embryos arising from the primary embryo (bar = 67 µm), (f) Histological sectioning of a 7-8 week old culture of secondary embryos of clone KKM 9. Arrow shows a secondary somatic embryo developed from the epidermal cells of the primary embryo (bar = 76 µm).

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Conversion to Plantlets

Somatic embryos proliferated from cotyledons (Figure 3A) and hypocotyls (Figure 3B) of primary somatic embryos. Somatic embryos longer than 10 mm and with a clear shoot and root were transferred to MGM medium for germination (Figure 3C). Once the plants developed a healthy shoot and root system (approx. two months after culture on MGM medium), they were transferred to soil and acclimatised to ex vitro conditions (Figure 3D). From our experience, the long culture history of the embryos did not affect the efficiencies of plant regeneration.

Ultrastructural Studies of Recurrent Embryogenesis

SEM studies revealed secondary embryos of globular and heart-shape arising from primary somatic embryos (Figure 3E). There is no synchronization as in somatic embryos of zygotic origin (Dos Santos and Machado, 1989). These secondary embryos were observed developed from the epidermal and sub-epidermal cells of the primary somatic embryos (Figure 3F). An epidermal cell origin of somatic embryos is more desirable for transformation than a mesophyll origin (Ellis, 1995, Raemaker, 1995). This is of particular importance for genetic transformation of the plant itself as those cells in the immature embryo that differentiate into embryogenic cultures are surface cells and therefore are more accessible to gene transfer by Agrobacterium or biolistics than embedded cells.

ACKNOWLEDGEMENT

The authors wish to thank the Ministry of Science, Technology and the Environment, Malaysia for financial support (IRPA Project No. 01-04-07-0301) and the Director General of Malaysian Cocoa Board for permission to publish these results. The technical assistance of Mohd. Firdaus and Jainab Madali is gratefully acknowledged.

REFERENCES

Alemano, L., Berthouly, M., Michaux-Ferrière, N. (1996). Histology of somatic embryogenesis from floral tissue cocoa. Plant Cell, Tissue and Organ Culture 46: 187-194. Dos Santos, A.V.P., Machado, R.D. (1989). A scanning electron microscope study of Theobroma cacao somatic embryogenesis. Ann. Bot. 64: 293-296. Ellis, D. (1995). Genetic transformation of somatic embryos. In Biotechnology in Agriculture and Forestry 30 : Somatic embryogenesis and synthetic seed I, Y.P.S. Bajaj ed. Springer-Verlag Berlin. pp 207-220. Hofer, M. (2004). In vitro androgenesis in apple - improvement of the induction phase. Plant Cell Rep. 22: 365-370. Kantharajah, A.S., Golegaonkar, P.G. (2004). Somatic embryogenesis in eggplant. Sci. Hortic. 99:107- 117. Kononowicz, H., Kononowicz, A.K., Janick, J. (1984). Asexual embryogenesis via callus of Theobroma cacao L. Z. Pflanzenphysiol Bd.113S: 347-358 Martenelli, L., Candioli, L., Costa, D., Poletti, V., Rascio, N. (2001). Morphogenic competence of Vitis rupestris S. secondary somatic embryos with a long culture history. Plant Cell Rep. 20: 279-284. Puigderrajols, P., Mir, G., Molinas, M. (2001). Ultrastructure of early somatic embryogenesis by multicellular and unicellular pathways in cork oak (Quercus suber L.). Ann. Bot. 87: 179-189. Raemakers, C.J.J.M., Jacobsen, E, Visser, R.G.F. (1995). Secondary somatic embryogenesis and applications in plant breeding. Euphytica 81: 93-107. Snedecor, G.W., Cochran, G.W. (1989). Statistical Methods, Iowa State University Press, Ames, United States of America. Söndahl, M.R., Liu, S., Bellato, C.M., Bragin, A. (1993). Cacao somatic embryogenesis. Acta Horticuturae 336: 245-248

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Toribio, M., Fernandez, C., Celestino, C., Martinez, M.T., San-Jose, M.C., Vieitez, A.M. (2004). Somatic embryogenesis in mature Quercus robur trees. Plant Cell Tissue & Organ Culture 76: 283-287. Vasic, D., Alibert, G., Skoric, D. (2001). Protocols for efficient repetitive and secondary somatic embryogenesis in Helianthus maximiliani (Schrader). Plant Cell Rep. 20: 121-125. Wood, G.A.R., Lass, R.A. (1987). Cocoa. Longman Scientific & Technical, Essex, England.

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SOMATIC EMBRYO GERMINATION AND CONVERSION INTO PLANTLETS IN COCOA (THEOBROMA CACAO L.)

Tan C.L. Cocoa Biotechnology Research Center, Malaysian Cocoa Board, Lot 4 - 6, Block B Sri Kemajuan Industrial Estate, Mile 6 ½, Tuaran Road, 88400 Kota Kinabalu, Sabah.

Malaysian Cocoa J. 1: 36-39 (2004) ABSTRACT Well-developed somatic embryos selected from a repetitive somatic embryo line derived from staminode tissues of immature flowers were used for germination and conversion studies. Glucose significantly improved the germination of somatic embryos as compared to maltose and sucrose. One-third strength Woody Plant Medium also improved the rate of germination of somatic embryos but high CO2 (20,000 ppm) did not affect the germination rate of the somatic embryos. Other factors such as addition of methyl laurate, activated charcoal and removing the cotyledons did not influence the rate of germination.

Key words: Somatic embryos, Germination, Conversion, Theobroma cacao

INTRODUCTION

Applications of biotechnology to plant improvement is frequently hampered by a lack of a reliable regeneration system from plant cells. Somatic embryogenesis is a useful system for genetic transformation not only because somatic embryos can be induced to repetitively undergo somatic embryogenesis, but also secondary embryos can be germinated into plants. In cocoa, the low efficiency of somatic embryo germination and conversion still remains a significant barrier affecting the development of a successful somatic embryogenesis system. In this study, the influence of medium salts, carbon dioxide, carbon source and other factors (cotyledon removal, activated charcoal, methyl laurate and calcium chloride treatments) on germination and conversion of somatic embryos were studied.

MATERIALS AND METHODS

Plant Materials - Well-developed somatic embryos (with at least 10 mm in length, Figure 1A) were selected from a repetitively somatic embryo line derived originally from staminodes (Tan and Furtek, 2003) of clone KKM 19.

Culture Medium and Physical Parameters - Embryos were then cultured on MGM medium (Figure 1B, C), consisting of 1/5 DKW salts, 5 gl-1 sucrose, 10 gl-1 glucose, 100 mgl-1 myo-inositol, 2 mgl-1 glycine, 2.2 gl-1 Phytagel (Sigma), pH 5.8, for a period of eight weeks in the light (with three subculture in between). The temperature was maintained at 25-26oC, humidity at 55-65% RH, and light at 1,850 lux (3,441 µE m- 2 -1 s ) from cool white fluorescent lamp. These conditions were maintained for both ambient and high CO2. Embryos were assessed for shoot, root and secondary embryo development after eight weeks. After approx. two months in culture, embryos that had developed sufficient roots and green leaves were transferred to ex vitro conditions (Figure 1D).

Experimental Conditions

Carbon dioxide (CO2) - Two treatments were carried out, one at ambient CO2 (approx. 450 ppm) and the other at high CO2 (20,000 ppm) inside a growth chamber equipped with light, temperature, humidity and carbon dioxide controls (Rubarth Apparate GmbH, Germany) Basal salts - Three treatments were carried out: 1/5 X DKW (Driver & Kuniyuki, 1984) basal medium, 1/3 X WPM (Lloyd & McCown, 1980) basal medium, and 1/3 X MS (Murashige & Skoog, 1962) basal medium.

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Carbon source - Four different treatments were carried out: MGM based medium: 3% glucose + MGM basal medium, 3% maltose + MGM basal medium, 3% sucrose + MGM basal medium.

Other factors - The following were also studied for their efficacy in improving the germination of somatic embryos: Forty-four (44) micromolar of lauric acid methyl ester (Sigma L-7272) in MGM, pretreatment with a supersaturated solution of calcium nitrate [Ca(NO3)2.4H2O, Sigma C-2536] and kept in the dark for three days prior to culture on MGM medium, removal of cotyledons, and addition of 0.5% activated charcoal (Sigma C-6289) to MGM.

Statistical Analysis - Each treatment consisted of at least five replicates (one somatic embryo per baby food jar). Means and standard errors (S.E.M.) were used throughout and statistical significance among values were assessed using ANOVA (Snedcor and Cochran, 1989) incorporating the post-hoc Tukey-Honestly significantly difference (Tukey HSD) test using commercial statistical analysis software (Minitab®). A probability of <0.05 was considered significant.

Figure 1. Germination and conversion of clone KKM 19 T. cacao somatic embryo: (A) somatic embryos longer than 10 mm in length (bar = 2.4 mm), (B) three-weeks old germinated somatic embryo (bar = 8.7 mm), (C) ten-week old converted somatic embryo cultured in a ventilated baby food jar (bar = 1.7 cm), (D) three-month old acclimatized ex vitro plant (bar = 8.3 cm).

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RESULTS AND DISCUSSION Carbon Dioxide

Figuera et al. (1991, 1993) reported improved shoot elongation and leaf, and root development of cotyledonary somatic embryos after treatment with 20,000 ppm CO2. In this study, CO2 did not influence the conversion of the cocoa somatic embryos into plantlets (Table 1). A secondary embryo developed from only one somatic embryo under ambient CO2.

Table 1. Influence of CO2 on conversion of Theobroma cacao somatic embryos

Treatment Total no. of Shoots only Roots only Both shoots and roots embryos tested (%) (%) (%) Ambient CO2 20 5.0 5.0 30.0 High CO2 (20,000 ppm) 14 7.1 0 21.4 Values are mean of at least five replicates. No values in any column are significantly different at the 95% confidence level.

Basal Salts

The highest percentage of germination (both shoots and roots) of somatic embryos occurred on 1/3 x WPM medium whereas no germination was observed on 1/5 x DKW or 1/3 x MS medium (Table 2). WPM medium has a low ammonium nitrate concentration as compared to the other two media (MS and DKW).

Table 2. Germination of somatic embryos on different basal medium

Treatment Total no. of Shoots only Roots only Both shoots and roots embryos (%) (%) (%) 1/5 X DKW medium (MGM) 9 0 22.2 0 1/3 X MS medium 6 0 50.0 0 1/3 X WPM medium 9 0 33.3 22.2 Values are mean of at least five replicates.

Carbon Source

The highest percentage of germination (both shoots and roots) was found in medium containing glucose only (33.3%, significant at P<0.05) (Table 3). MGM medium, which contains a combination of 20% glucose and 10% sucrose, was second with 20.0% of germination. Maltose and sucrose medium did not promote germination. Kononowicz and Janick (1984) also found that carbon source significantly influences the development of asexual embryos in cocoa, glucose being the most superior. Most somatic embryos produced roots only and few produced shoots only.

Table 3. Germination of somatic embryos on different carbon source

Treatment Total no. of Shoots only Roots only Both shoots and embryos (%) (%) roots (%) MGM medium 15 0 46.7 20.0 (20% glucose, 10% sucrose) 30% glucose medium 15 0 26.7 33.3*** 30% maltose medium 20 10.0 0 0 30% sucrose medium 15 6.7 13.3 6.7 Values are mean of at least five replicates. *** indicates significance at P<0.05.

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Methyl Laurate, Calcium Nitrate Dessication, Activated Charcoal, and Decotyledon

Methyl laurate, a methyl ester of lauric acids, is used for chemical pruning of field-grown crops. It also causes necrosis of the terminal bud and thus releases axillary buds from apical dominance. In hybrid rose, it promotes the development of shoots and roots in somatic embryos (Sarasan et al., 2001). However, addition of 44 µM methyl laurate to MGM medium did not improve the germination of somatic embryos. Pretreatment of somatic embryos in a supersaturated solution of calcium nitrate for three days prior to transferring to MGM medium did not improve the germination of the embryos. The somatic embryos shriveled and died due to excessive dehydration. Addition of activated charcoal, which is known to enhance root growth in some plants (Tang et al., 2000), did not influence the germination of somatic embryos. The removal of cotyledons from the somatic embryos resulted in the production of new shoots, but did not enhance germination as reported by Adu-Ampomah et al. (1988).

CONCLUSIONS

From our studies, glucose significantly improves germination (both shoots and roots elongation) of the somatic embryos of T. cacao compared to maltose and sucrose-containing media. One-third strength of Woody Plant Medium also enhances germination, but high carbon dioxide did not.

ACKNOWLEDGEMENTS

The authors wish to thank the Ministry of Science, Technology and the Environment, Malaysia for financial support (IRPA Project No. 01-04-07-0301) and the Director General of Malaysian Cocoa Board for permission to publish these results. The technical assistance of Mohd. Firdaus and Jainab Madali are gratefully acknowledged. Dr. Douglas Furtek is thanked for editing the manuscript.

REFERENCES

Adu-Ampomah, Y., Novak, F., Afzar, R., and Van Duhem, M. (1988). Determination of methodology to obtain shoot tip culture of cocoa. In Proc. 10th Int. Cocoa Res. Conf., Santo Domingo, Dominican Republic, 17-23 May, 1987. pp129-136. Driver, J.A. and Kuniyuki, A.H. (1984). In vitro propagation of paradox walnut rootstock. HortSci. 19:507-509. Figuera, A., Whipkey, A. and Janick, J. (1991). Elevated CO2 facilitates micropropagation of Theobroma cacao L. In Proc. Int. Cocoa Conf., 25-28 Sept. 1991, Kuala Lumpur, Malaysia. Figuera, A. and Janick, J. (1993). Development of nucellar somatic embryos of Theobroma cacao L. Acta Hort. 336: 231-237. Kononowicz, A.K., Janick, J. (1984). The influence of carbon source on the growth and development of asexual embryos of Theobroma cacao. Physiol. Plant. 61: 155-162. Lloyd, G. and McCown, B. (1980) Commercially feasible micropropagation of mountain laurel, Kalmia latiflora, by use of shoot-tip culture. In Proc. Int. Plant Prop. Soc. 30: 421-427. Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plantarum 15: 473-497. Sarasan, V., Roberts, A.V., Rout, G.R. (2001). Methyl laurate and 6-benzyladenine promote the germination of somatic embryos of a hybrid rose. Plant Cell Rep. 20: 183-186. Snedecor, G.W., Cochran, G.W. (1989). Statistical Methods, Iowa State University Press, Ames, United States of America. Tan, C.L. and Furtek, D.B. (2003). Development of an in vitro regeneration system for Theobroma cacao from mature tissues. Plant Sci. 164: 407-412. Tang, H., Ren, Z. and Krczal, G. (2000). Improvement of English walnut somatic embryo germination and conversion by dessication treatments and plantlet development by lower medium salts. In Vitro Cell. Dev. Biol. 36: 47-50.

Malaysian Cocoa Journal 45

DISPERSAL OF TRICHOGRAMMATOIDEA BACTRAE FUMATA NAGARAJA (HYMENOPTERA: TRICHOGRAMMATIDAE) AFTER RELEASES IN A MALAYSIAN COCOA FIELD

I. Azhar1 and G. E. Long2 1Malaysian Cocoa Board, Locked Bag 211, 88999 Kota Kinabalu, Malaysia 2Department of Entomology, Washington State University, Pullman, Wa 99164, U.S.A.

Malaysian Cocoa J. 1: 40-45 (2004) ABSTRACT Dispersal behavior of Trichogrammatoidea bactrae fumata Nagaraja (TBF) in a cocoa field following releases was studied based on trap catches using different colors and shapes of traps. More TBF were caught on green and square traps than on yellow and cylindrical traps. Green traps may resemble green pods while square traps had a greater surface area resulting in a higher catch. Dispersal of TBF on the first three days was limited. The distribution around the release site became more even with time as the TBF dispersed. Based on the observed dispersal distances, it is suggested that TBF release sites be placed 200 m apart. However, recommendation of optimal release distance requires further studies taking into consideration other factors such as TBF ecology and behavior, release density, CPB population dynamics and pod phenology.

Key words: cocoa, cocoa pod borer, biological control, egg-parasitoid, dispersal

INTRODUCTION

Egg parasitoids of the genera Trichogramma and Trichogrammatoidea have been widely used as biological control agents for many lepidopterous pests (Stinner, 1977). The parasitoids are mass-reared on fictitious hosts and released in the field at specific intervals. The efficacy of the releases varies and depends on a number of intrinsic and extrinsic factors, as outlined by Ridgway et al. (1981): 1) parasitoid density, 2) host egg density, 3) extent of searching area, 4) role of semiochemicals, 5) method of release, 6) parasitoid dispersal behavior, 7) parasitoid quality, 8) insecticide application in or around the treated area, and 9) parasitoid species or strain.

Trichogrammatoidea bactrae fumata Nagaraja (TBF), is an indigenous, polyphagous, egg parasitoid that was first recorded parasitizing cocoa pod borer (CPB), Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae) in Malaysia in 1982 (Lim, 1983). The parasitoid can be mass-reared on eggs of the rice moth, Corcyra cephalonica Stainton (Lepidoptera: Pyralidae). Attempts to control CPB by inundative releases of TBF have had limited success (Lim and Chong, 1987; Azhar et al., 2001). Experimental releases of TBF in three cocoa fields over a year have resulted in 34-52% parasitism of CPB eggs. Yet several plantations that were experimentally releasing TBF totally abandoned the procedure for economic reasons and the apparent ineffectiveness of TBF in suppressing CPB infestations. The ineffectiveness of TBF in these plantations might have been due to interference from regular chemical sprays, which have serious adverse effects on TBF populations (Azhar, 1992, 1995). In addition to the insecticides, dispersal from the plots in which they were released may limit the full realization of TBF’s potential. Therefore, understanding the dispersal behavior of TBF will enable researchers and cocoa growers to formulate a parasitoid-release program that will allow successful control in a reasonable time and with minimum cost.

This paper describes the dispersal behavior of TBF in a cocoa field following releases. It is based on trap catches, and compares the effectiveness of traps of different color and shape on catches of TBF.

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MATERIALS AND METHODS

Study Site

The study was conducted in Block 2 at the Cocoa Research Station, approximately 35 km northeast of Tawau, Sabah, Malaysia. Block 2 was an old clonal cocoa germplasm trial block, which consisted of more than 50 Fo clones that had been budded onto Amelonado rootstock in 1958. The clones comprised the following groups: GS (Grenada Selection), I (Indonesia), ICS (Imperial College Selection), LAFI, NA (Nanay), NGK (New Guinea Keravat), PA (Parinari), QH (Quoin Hill), UA (Upper Amazon), UIT (Unidentified Trinitario), and WA (West African). The clones were planted in rows ranging from 41- 65 trees each with no replication. The trees were spaced ca. 3.5 m within rows and ca. 3.5 m between rows. Several clones had fewer trees remaining because of mortality to some of the original trees. The block received regular management practices, but insecticide spraying and parasitoid release had not been carried out since 1985. Mature Amelonado cocoa fields surrounded the block.

Traps Used

Square and cylindrical traps were used in the studies. Square traps were intended to trap flying TBF while cylindrical traps, by virtue of being inserted over the pods were intended to trap host-seeking TBF attracted to the pods. Yellow and green manila cards were used to make equal number of traps. Green and yellow colors were chosen to resemble the colors of large green pods and ripe yellow pods. Color might serve as a search cue to the TBF. Square traps were made by cutting pieces of manila cards into 17 x 17 cm squares. Cylindrical traps were made by rolling 30 x 17 cm pieces of manila card, and stapling them into cylinders approximately 6 cm in diameter. Square traps were hung by a short wire from the fan branches. Cylindrical traps were inserted over and pinned to older pods on fan branches. Nontoxic glue was applied to both sides of the square traps but only to the external surface of the cylindrical traps.

Dispersal Studies and Comparison of Trap Catches

Insectary propagated TBF used in the study were obtained from the Department of Agriculture, Sabah. Parasitized host eggs on paper (28 x 21.5 cm) were placed inside a manila card cylinder (ca. 11 cm diameter), which was hung horizontally from a fan branch of the release tree. The cylinder had openings at both ends to allow the escape of emerging parasitoids.

TBF were released during March and August 1989 with an estimated numbers of 188,474 and 129,848 , respectively (assuming that two adults emerged from rice moth with a 1:1 male to female ratio). The block was sprayed with lindane ca. 1 wk prior to the first release of parasitoids to ensure that naturally occurring TBF were absent. Parasitoids were released on a tree in a corner of the block. To monitor TBF dispersal patterns, 4 sticky traps (2 squares - green and yellow; 2 cylindrical - green and yellow) were placed on trees at various distances from the release tree in south, southeast and east directions in the block. Traps were collected and replaced starting at 0700 h daily for 11 days, which was more than its longevity period and thus provide adequate time for TBF to disperse. TBF and other groups of insects were counted in the traps under a hand lens. Pre-release monitoring was initiated one day before the first release to determine existing population levels of TBF.

Statistical Analysis

TBF counts over the two release and monitoring periods were pooled and subjected to a two-way analysis of variance (SAS Institute, 1988) to examine any effect of trap shape and color on TBF catches. TBF counts were transformed with √(x + 1) to normalized variances. Because of the nonsignificant trap shape x trap color interaction, only the main effects were compared by the paired t-test. Untransformed values are presented in the results.

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Dispersal patterns of TBF were analyzed by plotting mean number of TBF per trap against trap distances for each day after release.

RESULTS

Trap Comparisons

More TBF (P < 0.05) were caught on square traps than on cylindrical traps (Table 1). At the same time, significantly more (P < 0.05) TBF were caught on green traps than on yellow traps. When individual cell means were compared (possible because F value trap shape x trap color interaction was 0.0553), the difference between the mean number of TBF caught on green square traps to that on cylindrical traps was small (0.02) but significant (P < 0.05).

Table 1. Average number of TBF collected on sticky traps of different types and colors over 2 release periodsa.

Trap color Trap shape Green Yellow Meanb Square 0.48 ± 0.04 0.42 ± 0.03 0.45 ± 0.02A Cylindrical 0.46 ± 0.04 0.28 ± 0.02 0.37 ± 0.02B Meanb 0.47 ± 0.03a 0.35 ± 0.02b aTotal number of traps sampled over 11 days for the two releases was 2548. bMeans followed by a different letter (lower and upper case) are significantly different (LSD, P < 0.05).

TBF Dispersal

Results for the dispersal of TBF in relation to the release point are presented in Figure 1. Few TBF (<0.03 TBF/trap/d) were caught in the field prior to TBF release in March (first release) and these bore no definite relation to the point of release (Figure 1). Similar patterns of TBF dispersal were observed after each release. Patterns of distribution of TBF caught on days 1, 2 and 3 after release were the same in all directions. More TBF were caught in the traps closer to the release point. The number decreased markedly with increasing distance. Their dispersal pattern gave a highly skewed distribution, especially for day 1 and 2 after release. Rarely were TBF caught at a distance of more than 150 m away from the release point on day 1. With the exception of the east direction for the March release [Figure 1( 2c)] the average number of TBF caught on traps located less than 10 m away from the release point were >6 per trap. The total number of TBF caught each day decreased as time passed. The TBF caught were spread fairly evenly by day 6 after release. By day 11 after TBF release, no definitive distribution was observed. The mean number of TBF caught was more or less equal at any distance from the release point. No TBF were caught further than 250 m from their release point.

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Figure 1. Dispersal patterns of TBF in Block 2 from two release dates (1 = March 1989, 2 = August 1989) in the south (a), southeast (b) and east (c) directions from release point. 0 indicates prerelease.

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DISCUSSION

Comparisons of TBF caught on traps of different colors and shapes suggest a number of inferences. That more TBF were caught on green traps suggests a color preference by the TBF. Although there are no reports of color preferences for Trichogramma species, several insect species are known to be attracted to a specific color. Yudin et al. (1987) trapped significantly more thrips on white than on colored traps. The cabbage root fly, Delia radicum (L.), was found to use leaf color in selecting variably suitable host plants and was trapped more in yellow and white traps (Vernon and Borden, 1983). The green color might have a reflectance similar to that of the commonly available older green pods, which are more likely to harbor freshly oviposited CPB eggs. However, this conjecture has to be validated in a laboratory experiment.

That more TBF were caught on square traps is probably due to their greater surface area, since TBF may get trapped on either side of the trap. TBF are more easily blown onto the traps by wind beneath the canopy. In contrast, sticky cylindrical traps on cocoa pods only trap TBF individuals going after the eggs that may be present on the pods sleeved over by the cylindrical traps. Wind velocity has been reported to be important in the dispersal of Trichogramma spp. (e.g., Schread, 1932; Kot, 1964; Hendricks, 1967). However, the relative wind velocities within the canopy and underneath the canopy and their effects on TBF movement need further study.

The trapping studies (Figure 1) suggest that substantial movement from the release point does not occur immediately following release, since no TBF were caught >100 m away on the first day. Stern et al. (1965) observed the dispersal of radioactively labeled Trichogramma semifumatum (Perkins) in cotton over distances up to 600 m in one day. The relatively large numbers of TBF caught near the release point several days after release may be due to staggered emergence from the parasitized rice moth eggs resulting in more or less continuous release for several days. Currently, staggered emergence of TBF is the common practice where rice moth eggs of various parasitized ages, multiplied in Morrison cabinet, are glued on a piece of paper for field releases (Azhar et al., 2001). The subsequent decline of TBF caught in this area is probably due to their dispersal into the field and mortality from many causes. The discovery of TBF at distances >200 m on the sixth day after release suggests the migration of TBF from neighboring fields, where TBF are released irregularly. Although TBF were present >200 m from release point, they occurred there at too low a density to adequately control the CPB.

TBF move in all directions, implying that movement from the release point is in random directions. Nonrandom movement of Trichogramma away from a release point was reported by Allen and Gonzalez (1974) where egg parasitization was higher at nearby sites than around the release point because of saturation effect of the parasitoids. Our results (Figure 1) suggest that the effective dispersal distance of TBF is 100 m. This suggests that establishing release sites at 200 m intervals may increase their efficiency. However, this figure is almost 20 times further than the current practice of releasing the TBF at four trees interval within row and at four rows interval or approximately 12 m between and within row (Alias Awang., Per. comm.). In addition, the distance between release points should vary with the TBF release density, CPB egg populations and pod phenology, and these aspects have to be further studied.

The dispersal rate is important but it is not the primary factor in making TBF an effective biological control agent for the CPB. Other aspects such as the individual quality, field mortality (natural or insecticide), numbers of TBF released, and good searching behavior as well as pest population dynamics are also important for effective suppression of the pest (Keller and Lewis, 1985). It is suggested that future studies on these aspects could improve the prospects for successful management of the CPB.

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ACKNOWLEDGEMENTS

We thank Roslan Saadi, Abd. Karim Ramli, Zailani Mohd Jamil, and Norhazazi Mohd Alimuddin (all formerly at MARDI Tawau) for their technical assistance. William J. Turner and Lynell K. Tanigoshi of the Department of Entomology, Washington State University (WSU), and Paul C. Schroeder of the Department of Zoology, WSU, kindly read and criticized the manuscript. This paper forms part of the MARDI-JPNS collaborative project on cocoa pod borer, and was supported in part by (1) the American Cocoa Research Institute (ACRI), and (2) the Washington State University’s College of Agriculture and Home Economics Agricultural Research Center: Department of Entomology Project 0355 -- Ecosystem Interactions of Economically-Important Insects.

REFERENCES

Allen, J.C. and Gonzalez, D. (1974). Spatial attack patterns of Trichogramma pretiosum around release sites compared with a random diffusion model. Environ. Entomol. 3: 647-652. Azhar, I. (1992). Factors affecting cocoa pod borer, Conopomorpha cramerella (Snellen) (Lepidoptera: Gracillariidae), and its egg parasitoid, Trichogrammatoidea bactrae fumata Nagaraja (Hymenoptera: Trichogrammatidae), in Malaysian cocoa. Ph.D. dissertation, Washington State University, Pullman. Azhar, I. (1995). An overview on the management of key insect pests of cocoa with major emphasis on the cocoa pod borer, Conopomorpha cramerella. The Planters 71: 469-480. Azhar, I., Alias, A. and Meriam, M. (2001). Research on the Cocoa Pod Borer in Malaysia. Proc. Incoped 3rd Int. Sem., 16-17 October 2000. Kota Kinabalu Sabah, Malaysia, p. 105-113. Keller, M.A. and Lewis, W.J. (1985). Movements by Trichogramma pretiosum (Hymenoptera: Trichogrammatidae) released into cotton. Southwest. Entomol. (Suppl.) 8: 99-109. Kot, J. (1964). Experiments in the biology and ecology of species of the genus Trichogramma West. and their use in plant protection. Ekol. Pol., Ser. A 12: 243-303. Lim, G.T. (1983). Trichogrammatoidea bactrae fumata Nagaraja (Hymenoptera: Trichogrammatidae) a new egg parasitoid of Acrocercops cramerella Snellen. MAPPS Newsl. 7 (Suppl.): 5-6. Lim, G.T. and Chong T.C. (1987). Biological control of cocoa pod borer by periodic release of Trichogrammatoidea bactrae fumata Nagaraja in Sabah, Malaysia, pp. 71-80. In P.A.C. Ooi, G.C. Luz, K.C. Khoo, C.H. Teoh, M. Md. Jusoh, C.T. Ho and G.S. Lim (eds.), Management of the cocoa pod borer. Kuala Lumpur: Malaysian Plant Protection Society (MAPPS). Ridgway, R.L., Ables, J.R., Goodpasture, C. and Hartstack, A.W. (1981). Trichogramma and its utilization for crop protection in the U.S.A., pp. 41-48. In J.R. Coulson [ed.], Proceedings of the joint American-Soviet conference on use of beneficial organisms in the control of crop pests. College Park: Entomological Society America. SAS Institute. (1988). SAS/STAT User's Guide. Rel. 6.03 ed. Cary, N.C: SAS Inst. Inc. 1028 pp. Schread, J.C. (1932). Behavior of Trichogramma in field liberations. J. Econ. Entomol. 25:370-374. Stern, V.M., Schlinger, E.I. and Bowen, W.R. (1965). Dispersal studies of Trichogramma semifumatum (Hymenoptera: Trichogrammatidae) tagged with radioactive phosphorous. Ann. Entomol. Soc. Am. 58: 234-240. Stinner, R. E. (1977). Efficacy of inundative releases. Annu. Rev. Entomol. 22: 515-531. Vernon, R. S. and Borden, J.H. (1983). Spectral specific discrimination by Hylemya antiqua (Meigen) (Diptera: Anthomyiidae) and other vegetable-infesting species. Environ. Entomol. 12: 650-655. Yu, D.S.K., Laing, J.E. and Hagley, E.A.C. (1984). Dispersal of Trichogramma spp. (Hymenoptera: Trichogrammatidae) in an apple orchard after inundative releases. Environ. Entomol. 13: 371-374.

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EFFICACY OF MATING DISRUPTION USING SYNTHETIC SEX PHEROMONE FOR THE MANAGEMENT OF COCOA POD BORER, CONOPOMORPHA CRAMERELLA (SNELLEN) (LEPIDOPTERA: GRACILLARIIDAE).

Alias A. 1, W. Sadao2 and E.B. Tay1 1Malaysian Cocoa Board, Locked Bag 211, 88999 Kota Kinabalu, Sabah, Malaysia 2Japan International Research Center for Agricultural Sciences MAFF, Ohwashi 1-2, Tsukuba, Ibaraki 305, Japan

Malaysian Cocoa J. 1: 46-52 (2004) ABSTRACT The feasibility of using synthetic sex pheromone as a mating disruption agent for the control of the cocoa pod borer, Conopomorpha cramerella was examined by dispensing a 88:12 mixture of (E,Z,Z) - and (E,E,Z) - 4,6,10- hexadecatrienyl acetates in a 1.7 hectare cocoa (Theobroma cacao) field at the Cocoa Research Station, Quoin Hill, Tawau, Sabah, Malaysia. Attraction of male moths to sex pheromone traps in the treated field was completely inhibited for the first one month, but the catches were gradually increased thereafter. The data suggest that mating between sexes was disrupted at least in the first one month. Mating ratio of the females captured with sweeping net in the first month was reduced to 80% and 90% in the central and peripheral plots of the treated field, respectively, in contrast to the untreated field, which was 99%. Mean numbers of spermatophore in the female were reduced to 0.98 and 1.28 in the central and peripheral of the treated field respectively as compared with 2.01 in the untreated field. However, mating rates of cocoa pod borer moths and mean number of spermatophore per female increased thereafter. Although reduction of mating ratio and mean spermatophore indicated that mating chance was certainly reduced in the treated field, oviposition densities on cocoa pod were not significantly reduced. Therefore, it is concluded that the effect of mating disruption was insufficient to reveal a good control effect on field population, may be because of insufficient concentration of pheromone in the air and or the population of cocoa pod borer was higher in the cocoa field.

Key words: Cocoa, Cocoa pod borer, Synthetic sex pheromone, Mating disruption

INTRODUCTION

Cocoa pod borer, Conopomorpha cramerella (Snellen) has been the most important cocoa pest in Malaysia since its discovery in 1980 (Lim et al., 1982; Tay, 1987). It causes serious losses, amounting to 20-50% of the crop (Mumford, 1984). The measures for the control of cocoa pod borer have been reviewed (Ooi et al., 1987). Of the existing methods, chemical treatment is the most important and reliable method, but there are considerable disadvantages including the possibility of inducing resistance. Therefore, the development of alternative control measures is necessarily.

The female sex pheromone of C. cramerella was firstly isolated and synthesized by Beevor et al., (1984) and followed with field experimentation in Tawau, Sabah. The sex pheromone produced by the female moths has been shown to consist of at least five components. A mixture of the five components in 40:60:4:6:10 ratio was the most attractive to male moths (Beevor et al., 1984). Therefore, controlling this insect by means of mating disruption between male and female moths to prevent mating was possible. Preliminary 'confusion' trials carried out in BAL Plantations (Ho et al., 1987) and the Department of Agriculture, Tenom, Sabah (Tay and Sim, 1989) provided some evidence on the potential of this approach. However, the pheromone dispenser used had short longevity. With the new type of pheromone dispenser, we hope it could last for more than three months. The present study was therefore aimed to evaluate the performance of new type of pheromone dispenser and its effect on mating disruption of the cocoa moths.

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MATERIALS AND METHODS

Experiment Area - The experiment was conducted in two areas in the Cocoa Research Station, Quoin Hill, Tawau in February 1993. Each plot consisted of 1,936 trees (ca. 2 hectare). The distance between two plots was about 4 km. The first plot was treated with synthetic sex pheromone while the second plot served as a control. There was no chemical spraying during the duration of the experiment.

Synthetic Sex Pheromone - The dispensers used consisted of sealed polyethylene tubes 20 cm long and an aluminum wire. Each dispenser contained 60 mg of a 85:12:3 mixture of (E,Z,Z)- and (E,E,Z) -4,6,10- hexadecatrienyl acetate (E4Z6Z10-16:Ac and E4E6Z10-16:Ac) and (E,Z,Z) -4,6,10-hexadecatrien-l-ol (E4Z6Z10-16:OH) and 3 mg of chemical stabilizer. The major impurity in E4Z6Z10-16:Ac and E4E6Z10- 16:AC was the geometric isomers whose content was ca. 4.0% (2.7 mg/dispenser). One thousand nine hundred and thirty six (1,936) dispensers were set in the cocoa field at the rate of one dispenser per cocoa tree. The dispensers were hung on small twigs at 2.0-2.5 m above the ground.

Sex Pheromone Trap - Eight sex pheromone traps (SE. Trap, Sankei Chemical Company Limited, Kagoshima, Japan) were set at 2.5-3.0 m above ground at density of four traps per plot. Each trap was baited with rubber septum impregnated with 0.40:0.60:0.083:0.015:0.10 mg of E4Z6Z10-16:Ac; E4E6Z10- 16:Ac; E4Z6Z10-16:OH; E4E6Z10-16:OH and hexadecan-l-ol (16:OH) respectively. The traps were lined with glue to catch the moths. The distance between each trap was about 30 m. Observations were made daily for the first month and twice a week for the following months. The number of male moths caught in the trap was recorded. Sticky plates were cleaned regularly. Rubber septum was changed at intervals of two months.

Sampling Plot - Twelve sampling plots, consisting of 5 x 5 cocoa trees, were selected in the treated and untreated field. Twenty-five mature green pods were harvested weekly and the number of eggs on each pod was observed. The eggs were incubated in the laboratory to examine their hatchability.

Capturing Female Cocoa Pod Borer - Sweeping nets were used to capture the adult of cocoa pod borer. This was done by sweeping underneath the horizontal cocoa branches at weekly interval.

Longevity of Pheromone-Dispenser - Thirty pheromone dispensers were also set in the cocoa field to examine their longevity. Three pheromone dispensers were collected at 0, 1, 2, 3, 4, 6 and 8 months after exposure in the field. The dispensers were sent to Specialty Chemical Research Center of the Shin’Etsu Chemical Company Limited for analysis.

RESULTS Effect of Mating Disruption

C. cramerella moths were captured with sex pheromone traps throughout the treatment period as shown in Figure 1. The results indicated that mating between sexes was disrupted at least four weeks after the pheromone dispensers were placed in the cocoa field. However the number of male moth catches increased gradually thereafter. Therefore, the data suggest that the effective period of mating disruption was in the first month after treatment.

The results on the dissection of the female moths captured with sweeping net are shown in Figure 2. The mating rate of cocoa moths in the treated field was certainly reduced as compared to the untreated field. The data indicated that mating rate of cocoa moths captured from untreated field in the first month was 99% with mean spermatophore per female of 2.01. In contrast, the cocoa pod borer mating rate in central and peripheral plots of the treated field were 80% and 90% with mean spermatophore per female of 0.98 and 1.28 respectively. Generally, the mating rate and the mean number of spermatophore increased and fluctuated throughout the treatment period but still lower as compared to the untreated field.

Malaysian Cocoa Journal 53

40 Treatment Period 35

30 F42M F42C F20 25

20

Catch/trap 15

10

5

0 0 4 812162024 Weeks

Figure 1. Number of male moths caught in the treated (F42M and F42C) and untreated field (F20). [M = margin of the plot; C = center plot]

120

100

80

60

40 Mating rate (%)

20 F42M F42C F20 0 0 4 812162024

Weeks Figure 2. Mating rate of adult moths. [F42M = margin of the treated plot; F42C = center of the plot; F20 = untreated field]

Malaysian Cocoa Journal 54

Effect on Egg Density of C. cramerella

The mean densities of cocoa pod borer eggs in the treated and untreated field after treatment are shown in Figure 3. No significant reduction of egg densities was observed in the treated plots. The egg densities seem to fluctuate within the treatment period. However, the mean egg density in the peripheral plots (1.04 eggs/pod) was slightly higher as compared to the central plots (0.88 eggs/pod) in the first month after treatment. This suggests that the population of cocoa moths was higher in the peripheral than the central plots. Hence the moths could mate by chance. The results appear to correspond with the mating rate.

Low egg density in the untreated field (0.51 eggs/pod) could be associated with crop phenology. In Tawau, cocoa has two peak crops, April to May and October to November. Higher number of pods was observed in the untreated field as compared to the treated field during the treatment periods.

2.0

1.5

1.0 Mean eggs/pod 0.5

0.0 1 5 9 13 17 21 Weeks

Figure 3. The number of eggs observed on cocoa pod after treatment. [F42M = margin of the treated plot; F42C = center of the plot; F20 = untreated field]

Figure 4 shows the eggs collected in both treated and untreated field for observation in the laboratory for hatchability. Reduction in egg hatchability was observed in the central plots (54%) of treated field as compared to peripheral plots (67%) of the treated and untreated field particularly in the first months after treatment. Generally, the percentage of egg hatchability was higher in the untreated field (73%) as compared to the treated field. However, the percentage of egg hatchability increased and fluctuated thereafter for all treatment.

Malaysian Cocoa Journal 55

120 F42M F42C F20 100

80

60

40 Hatchability (%)

20

0 1 5 9 13 17 21 Weeks

Figure 4. Percentage of hatched eggs observed on the cocoa pod after treatment. [F42M = margin of the plot; F42C = center of the plot; F20 = untreated plot]

Migration of Mated Females

Although the mating disruption of C. cramerella was successful in reducing the mating rate, the reduction of population density was unclear because of the possibility of migration of mated females from the surrounding untreated field. If the mated females had migrated into the treated area, the density of eggs would have been higher in the peripheral plots of the treated field. However, there was no significant difference in the eggs densities between peripheral and central plots (Figure 3). This indicates migration of mated female moths was considered negligible in the experiment or there was migration but the control effect was unclear because of the size of treated field is rather small.

Longevity of Pheromone-Dispenser

Results of chemical analysis of cocoa pod borer pheromone dispenser after exposure in the cocoa field are shown in Table 1. For the improved formulation (brown type), no abnormal degradation was observed after 242 day’s exposure in the field. The weight and remaining active ingredients were decreased proportionately, which indicated no polymerization took place in the dispenser tube. The data also suggested that there was no significant change in the EZZ/EEZ ratio which indicated no isomerization from EZZ-isomer and EEZ-isomer took place. Based on this analysis, it seems that the effective periods of the new brown type dispenser is estimated to be 8 months after treatment. However, the results do not seem to correspond to the trap data in the pheromone-treated field and mating ratio.

Malaysian Cocoa Journal 56

Table 1. Contents of cocoa pod borer pheromone dispenser exposed in cocoa field.

Date of Days Total Pheromone amount in dispenser (mg) Estimated recovery content release rate (mg) EZZ EEZ EZZ-OH Total (mg/month)

15 Feb 93 0 67.0 53.0 8.2 1.8 63.0 8.8 15 Mac 93 28 61.1 45.8 7.5 1.5 54.8 4.9 21 Apr 93 65 52.2 40.4 6.8 1.5 48.7 9.1 15 May 93 90 45.1 33.6 5.8 1.7 41.1 6.5 15 Jun 93 120 37.6 28.3 4.9 1.4 34.6 5.7 15 Aug 93 181 27.3 18.4 3.3 1.4 23.1 3.4 15 Oct 93 242 19.6 12.6 2.3 1.3 16.2

DISCUSSION

Disruption of pheromonal communication between the sexes of many species of Lepidoptera can be effected by permeating the air with synthetic compounds identical or similar to the natural pheromone of the target species (Roelofs and Carde, 1977, Shorey, 1977, Wakamura, et al., 1989). In the present study, we found that mating disruption of cocoa pod borer can be achieved by dispensing an 88:12 mixture of (E,Z,Z) - and (E,E,Z) - 4, 6, 10 - hexadecatrienyl acetates into the atmosphere of the cocoa field, although it is only for a short period. Reduction in mating rate and mean number of spermatophore per female was observed in the treated field but our observation did not correspond with oviposition densities. High percentage of egg hatchability also suggested that mating disruption did not result in a good control of cocoa pod borer population. This may indicated that the amount of the chemical permeated through the air was insufficient to confuse the male moths or the wind may cause dilution of pheromone in the air. As high aerial concentration of pheromone is needed to obtain a good control effect (Wakamura, 1992), it is therefore necessary to understand the relationship between pheromone concentration in the air and mating disruption to obtain optimum response.

There is also a possibility of migration of mated female moths since the size of treated field is rather small, although the moths were reported as weak flyer (Lim et al., 1982). Migration may occur when the moths flutters among cocoa trees. The results showed that the mean egg density in the peripheral plots was slightly higher as compared to the central plots of treated field, although there are not significantly different. This may suggested that there was migration of mated female moths from the surrounding untreated field.

Effective control is also believed to be higher when the population density is low and the effect tends to disappear when population become high. This may explain why the trap data did not correspond to the longevity of pheromone dispenser. Perhaps the population of cocoa pod borer increased after treatment.

The above study demonstrates the potential use of synthetic sex pheromone as mating disruption agent for the management of cocoa pod borer although the control effect was regards insufficient. More research on technical aspects and cost effectiveness will be required before commercial application.

Malaysian Cocoa Journal 57

ACKNOWLEDGMENTS

This work formed part of a joint collaboration project between Malaysian Cocoa Board and the Tropical Agriculture Research Center, Japan (currently Japan International Research Center for Agricultural Sciences). We would like to thank Dato' Dr. Azhar Ismail, the Director General of Malaysian Cocoa Board for his permission to present this paper, Dr. Lee Ming Tong, Fellow Research for his constructive comments and Dr. C.L. Bong, Ex. Senior Research Officer of Malaysian Cocoa Board for kindly reading through the manuscript. Appreciation is due to Shin’Estu Chemical Company Limited, Japan for supplying the pheromone dispensers. We are grateful to Dr. H. Suzuki and his staff for the assistance in chemical analysis of cocoa pod borer pheromone dispenser. We would also like to thank the Director of the Department of Agriculture, Sabah for providing the trial plots for the studies. Lastly we also thank Mr. Mohd Azmi Sunda, Assistant Research Officer of Malaysian Cocoa Board for his technical assistant during the course of the trial.

REFERENCES

Beevor, P.S., Day, R.K., and Mumford, J.D. (1984). Female sex pheromone of cocoa pod borer moth, Acrocercop cramerella: Identification and field evaluation. In cocoa and coconuts: Progress and outlook [eds. Puspharajah, E. and Chew, P.S.] pp287-292, Incorporated Society of Planters, Kuala Lumpur. Ho, C.T., Beevor, P.S., and Mumford, J.D. (1987). A practical approach to the control of the cocoa pod borer moth using synthetic sex pheromone in an integrated system. In Management of the cocoa pod borer. (Edited by Ooi et al., 1987). Malaysian Plant Protection Society, Kuala Lumpur. Lim, G.T., Tay, E.B., Pang, T.C. and Pan, K.Y. (1982). The biology of cocoa pod borer Acrocercops cramerella Snellen and its control in Sabah, Malaysia. Proc. Int. Conf. Pl. Prot. in Tropics. pp: 275- 287. Mumford, J.D. (1984). Control of the cocoa pod borer (Acrocercops cramerella): A critical review. In International Conference on Cocoa and Coconut, 15-17 October, 1984. Ooi, P.A.C., Chan, L.G., Khoo, K.C., Teoh, C.H., Jusoh, M.M., Ho, C.T. and Lim, G.S. (1987). Management of the cocoa pod borer. Malaysian Plant Protection Society, Kuala Lumpur. 192pp. Roelofs, W.L., and Carde, R.T. (1977). Responses of Lepidoptera to synthetic sex pheromone chemicals and their analogues. Annu. Rev. Entomol. 22: 377-405 Shorey, H.H. (1977). Manipulation of insect pests of agricultural crops, pp. 353-367. In H.H. Shorey and J.J. McKelvey [eds.] Chemical control of insect behavior: theory and application. Wiley. New York. Tay, E.B. (1987). Control of cocoa pod borer - The Sabah Experience. In Management of the cocoa pod borer. (Edited by Ooi et al., 1987). Malaysian Plant Protection Society, Kuala Lumpur. Tay, E.B. and Sim, C.H. (1989). Field evaluation of a synthetic sex pheromone of the control of cocoa pod borer (Conopomorpha cramerella Snellen). MAPPS Newsletter 13(2): 25-28. Wakamura, S., Takai, M., Kozai, S., Inoue, H., Yamashita, I., Kawahara, S., and Kawamura, M. (1989). Control of the beet armyworm, Spodoptera exigua (Hubner) (Lepidoptera: Noctuidae), using synthetic sex pheromone. I. Effect of communication disruption in Welsh Onion Fields. Appl. Ent. Zool. 24 (4): 387-397. Wakamura, S. (1992). Development in application of synthetic sex pheromone to pest management. Japan Pesticide Information. No. 61.

Malaysian Cocoa Journal 58

QUALITY ASSESMENT OF COCOA BEANS PRODUCED BY SMALLHOLDERS FROM DIFFERENT REGIONS IN MALAYSIA

Hii C. L., Y. K. C. Samuel and I. Nor Haslita Cocoa Downstream Research Center, Malaysian Cocoa Board, Lot 3, Jalan P/9B Seksyen 13, 43650 Bandar Baru Bangi, Selangor Darul Ehsan, Malaysia

Malaysian Cocoa J. 1: 53-58 (2004) ABSTRACT The objective of this study is to assess the quality of cocoa beans produced by smallholders from different regions in Malaysia, such as Sabah, Sarawak, Perak, and Pahang. Most of the samples were obtained from beans fermented in plastic sacks, with a fermentation duration of 5 days and dried using natural technique in 4-5 days. Results showed no significant difference (p>0.05) among the bean samples for moisture content, pH, degree of fermentation (cut test score and fermentation index), and sensory evaluation (cocoa, bitterness, astringency, and sourness flavour attributes) based on the different locations. Cut tests showed that all the samples were well-fermented with percent brown beans of more than 60% which agreed well with the fermentation index.

Key words: Cocoa, Drying, Fermentation, Smallholders, Quality

INTRODUCTION

The quality of cocoa beans is determined by various pre- and post-harvesting factors such as varieties, soil, climate, harvesting, fermentation, drying, and storage. Fermentation and drying play a very significant role as flavour precursors are formed during these processes and the compounds formed will later react with each other during roasting to produce the typical chocolate flavour (Jinap et al., 1995). In Malaysia, various processing methods are used and this could result in cocoa beans of various qualities being produced. Currently, smallholders dominate the production of cocoa beans in Malaysia with national planting hectarage of more than 62% (Anon, 2002).

The quality attributes of cocoa beans can be determined from cut tests and sensory evaluation. The visual assessment in cut test provides information on the degree of fermentation, whereas flavour quality can be determined through sensory evaluation. Consumers generally dislike chocolate with excessive bitterness, astringency, sourness, and other unacceptable off flavours. Useful information can also be obtained from moisture content and pH due to its influence on storage, processing (e.g. alkalization and roasting), and end product quality. Therefore, information that relates to the bean’s attributes enable manufacturers and even farmers to adjust the processing parameters to achieve a better quality product.

The objective of this study is to assess the quality of cocoa beans produced by smallholders from different regions in Malaysia, such as Sabah, Sarawak, Perak and Pahang.

MATERIALS AND METHODS

Cocoa Bean Samples - Cocoa bean samples were obtained from smallholders in Sabah, Sarawak, Pahang, and Perak through the Licensing and Grading Unit in the respective areas. At least four samples (weighed 2 kg per sample per smallholder) were obtained from each region. A survey form was also distributed to smallholders to obtain information on processing such as fermentation and drying methods. A total of 18 samples were collected. Five samples each were collected from Sarawak and Perak while four samples each were collected from Pahang and Sabah.

Bean Moisture Content - Moisture content of the beans was determined according to AOAC (1990). Cocoa beans were peeled by hand and the resulting nibs (10 g) were ground using an electric blender (Waring,

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USA). Two grams of the ground sample was weighed using an analytical balance (Sartorius, Germany) and dried in an aluminium dish using an air-ventilated oven (Memmert, Germany) at 105°C until constant weight was achieved. This measurement was taken daily in triplicate.

pH - The pH was determined according to AOAC (1990). Ground nibs (5 g) were homogenised in 45 ml boiled distilled water. The homogenate was filtered with Whatman No.4 filter paper and cooled to 20-25oC. pH was determined using a pH meter (Metler Toledo, USA) which had been calibrated with buffers at pH 4 and 7.

Cut Test - This was carried out according to the Malaysian Standard MS 293 (Anon, 1995). Three hundred pieces of dried cocoa beans were cut lengthwise through the middle using a penknife. Both halves of each bean were examined in full daylight, and the beans were classified as fully brown, purple-brown, fully purple or slaty. The percentage count of each colour class was calculated for the Cut Test Score (CTS) as below:

CTS = (10 x % fully brown) + (5 x % partly purple-brown) + (0 x % fully purple & slaty)

Fermentation Index - This was determined according to the method of Gur’eva and Tserevitinov (1979). Ground cocoa nib samples of 0.5 g were added to a 50 ml mixture of methanol and HCl at a volume ratio of 97:3 and homogenized. The mixture was left in the cold room at less than 8°C for 16-18 hours and filtered using Whatman No.1. The filtrate was collected and the absorbances at 460 nm and 530 nm were measured spectrophotometically.

Sensory Evaluation of Cocoa Liquor - Five hundred grams of cocoa beans was processed using a laboratory winnower and breaker (John Gordon, England) to obtain cocoa nibs. The nibs were roasted in an oven (Memmert, Germany) at 140°C for 35 minutes and cooled to room temperature. The roasted nibs were then ground in a laboratory mortal and pestle mill (Pascal Engineering, England) for three hours to obtain cocoa liquor. Sensory evaluation of cocoa liquors was conducted by trained sensory panels from Malaysian Cocoa Board. West African cocoa liquor obtained from a local factory was used as a reference.

Statistical Analyses - The data were analysed for one-way ANOVA using SAS statistical software (Version 8, SAS Institute, USA) at 95% confidence level.

RESULTS AND DISCUSSION Survey

Malaysian cocoa smallholders farm size generally varies according to region. For instance in the Peninsular and Sarawak about 86% and 92% of the smallholders, respectively, have farm size of 2.9 ha and below while in Sabah only 57% of smallholders belong to this scale of farm size.

Results of the survey (Figure 1) showed that most of the bean samples were obtained from plastic sack fermentation (62.5%) followed by box fermentation (25%). The remaining samples (12.5%) were obtained from a combination of techniques such as plastic sack fermentation initially followed by either box or basket fermentation. Plastic sack fermentation has been used by the smallholders because it is easier to mix, simply by pressing the sacks on the ground, and that the material is cheap and easily available. The plastic sack can be filled with wet cocoa beans ranging from 30 to 90 kg. Different fermentation techniques could influence end product quality (Lopez and Dimicks, 1991). In terms of fermentation duration, most of the samples were obtained from 5-day fermentation (56.3%) followed by 6-day fermentation (25%). The remaining samples were obtained from 4-day (6.3%) and 3-day fermentation (12.5%).

The survey (Figure 1) also showed that the samples were dried mostly using natural technique (87.5%), while the remaining samples were artificially dried (6.3%) or dried using combined techniques

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(6.3%). Naturally dried cocoa beans have been reported to have better flavour quality as compared to artificially dried beans due to the gentle drying process (Jinap, 1995). Dissipation of acetic acid is more efficient in natural drying as compared to the faster artificial drying where entrapment of acids causes high acidic flavour in cocoa beans (Duncan et al., 1989). In terms of drying duration, most of the samples were obtained from 4-day (37.5%) and 5-day drying (31.3%). This was anticipated as most of the samples were dried using the natural technique which is weather dependent and usually requires longer drying time. In some samples the drying duration was even 6 days (18.8%) due to the similar reasons. Only a small proportion of samples were obtained from 3-day drying (12.5%), which could be from artificial drying.

% 100 Fermentation Drying

90 87.5

80 70 62.5 60 56.25

50

40 37.5 31.25 30 25 25 18.75 20 12.5 12.5 12.5 10 6.25 6.25 6.25

0 l l s s s s s s s s s ck ra

Box her tu 3 day 4 day 5 day 6 day 3 day 4 day 5 day 6 day Ot Na mbined Artificia o stic sa

C Pla Figure 1. Fermentation and drying techniques survey summary

Moisture Content and pH

Cocoa beans are generally dried to moisture content of less than 7.5% for the purpose of safe storage. Insufficiently dried beans are normally associated with mould formation which can lead to development of off-flavour, increase in free fatty acids and production of mycotoxins. In downstream processing, the moisture content will influence plant operating conditions, as more moisture will need to be removed in order to produce a consistent quality end product. Moisture inside the beans would also affect chemical reactions during alkalization and roasting.

Moisture content of the dried beans samples (Table 1) showed values ranging from 7.15% to 8.27%. The results showed that samples obtained from Sarawak and Pahang were well below the safe level (7.5%). Samples from Perak and Sabah were slightly under-dried since most of the samples were dried naturally. Cocoa beans would have difficulty attaining safe moisture content if weather conditions were not conducive for natural drying. Statistically, there was no significant difference among the moisture contents of the bean samples (p>0.05) based on different locations.

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Table 1. Moisture content and pH of dried cocoa beans samples

Source Attributes Sarawak Perak Pahang Sabah p-value Moisture 7.15a 8.27 a 7.20 a 8.18 a 0.11 content (%) pH 5.96 a 5.98 a 5.42 a 5.91 a 0.50 Mean values having a common letter within the same row are not significantly different at the 95% confidence level (p>0.05).

Measurement of pH showed values ranging from 5.42 to 5.98 as indicated in Table 1. The pH values were mostly at the less acidic level (pH > 5.50) except for the Pahang samples which were at the medium level (pH 5.20-5.49). The pH values recorded were much less acidic than those reported by Jinap et al. (1995) between 4.64 and 4.85 for Malaysian cocoa beans. Highly acidic beans are associated with pH of less than 5.2, and the best flavoured West African beans usually have pH values around 5.5 (Jinap, 1994). Highly acidic beans are associated with low chocolate flavour, possibly due to over degradation of storage proteins. Jinap et al. (1995) reported that chocolate made from medium pH beans received a higher response in strong chocolate flavour than those made from low and high pH beans. Statistically, there was no significant different among the pH of the bean samples (p>0.05) based on the different locations.

Cut Test and Fermentation Index

Results of the cut test are as shown in Figure 2. Bean surface colour of unfermented beans are generally slaty, underfermented beans are purple, and fully fermented beans are brown in colour (Shahrir and Mamot, 1987). The quantity of slaty and purple beans was higher in the Sarawak and Pahang samples as compared to the Perak and Sabah samples. The presence of these beans in high amounts is not desired as unfermented and underfermented beans tend to introduce excessive bitterness and astringency in the finished products. Astringency and bitterness flavours are mostly due to the presence of polyphenols that are not oxidised during fermentation and drying (Misnawi et al., 2000). In terms of percent purple-brown beans the values were at a reasonably accepted level (5.9%-18.1%). In general, all samples were well- fermented with percent brown beans of more than 60%.

% 100 90 80 74.63 72.30 71.75 70 60.20 60 50

40

30

18.10

20 17.25 13.00 10.88 10 9.63 5.90 5.13 4.00 0 Sarawak Pahang Perak Sabah Source

Purple+slaty Purple-brown Brown

Figure 2. Cut test results of dried cocoa beans samples

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The results were quite consistent with cut test scores and closely agreed with the fermentation index (fully fermented beans, CTS > 600 and FI > 1) as shown in Table 2. This was anticipated since more than 80% of the samples were fermented for 5-6 days according to the survey. Anthocyanins which are responsible for the purple colour of the beans, generally decreased by 93% during the course of fermentation (Cros et al., 1984). Statistically, there was no significant difference among the bean samples (p>0.05) in terms of cut test score and fermentation index based on different locations. This showed good consistency among the attributes.

Table 2. Degree of fermentation of dried cocoa beans samples

Source Attributes Sarawak Perak Pahang Sabah Cut test score (CTS) 692.50 a 752.50 a 794.38 a 803.75 a Fermentation index (FI) 0.98 a 1.35 a 1.07 a 0.91 a Mean values having a common letter within the same row are not significantly different at the 95% confidence level (p>0.05).

Sensory Evaluation

The flavour scores of cocoa liquor for various attributes are as shown in Table 3. Statistically, there was no significant difference among the bean samples (p>0.05) in terms of cocoa, bitterness, astringency, and sourness attributes since most of the samples were fermented and dried in a similar manner. This ensures that microbial and enzymatic reactions progressed similarly.

However, the samples were lower in cocoa flavour and higher in bitterness, astringency, and sourness as compared to the West African reference sample. It must be noted that the reference sample was obtained from a commercial roasting system, as compared to laboratory prepared samples using the oven method. Nonetheless, improvement of bean flavour quality could be achieved through various post-harvest treatments for Malaysian cocoa beans (Meyer et al., 1989; Biehl et al., 1990; Duncan et al., 1989; Hii, 2002).

Table 3. Flavour score of dried cocoa beans samples in sensory evaluation

Source Flavour Sarawak Perak Pahang Sabah p-value attributes Cocoa (7) 4.51 a 4.51 a 4.39 a 4.27a 0.91 Bitter (3) 3.36 a 3.41 a 3.44 a 3.51 a 0.91 Astringent (2.5) 3.69 a 3.81 a 3.50 a 3.84 a 0.53 Sour (1.5) 2.41 a 2.68 a 2.63 a 2.53 a 0.25 Mean values having a common letter within the same row are not significantly different at the 95% confidence level (p>0.05). Values inside bracket indicate the score of the reference sample.

CONCLUSION

Assessment of some quality attributes of bean samples has provided important information on the current quality of Malaysian cocoa beans produced by smallholders. Most of the samples were obtained from beans fermented in plastic sacks, with a fermentation duration of five days and dried using natural technique in 4-5 days. Some cocoa bean samples from Perak and Sabah had a moisture content of more

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than 7.5%. Most of the bean samples were at the less acidic level (pH range 5.91-5.98) except for the Pahang samples which were at the medium level (pH = 5.42). Cut tests showed that all samples were well- fermented with percent brown beans of more than 60%. Results were quite consistent with cut test scores (Score range 692.5-803.75) and agreed closely with the fermentation index (Index range 0.91-1.35). Sensory evaluation showed that the samples were lower in cocoa flavour and higher in bitterness, astringency, and sourness as compared to the West African reference sample. Results also showed no significant difference (p>0.05) among the bean samples based on the different locations in any of the quality attributes assessed.

REFERENCES

Anon. (2002). Malaysian Cocoa Monitor. 11(2) December 2002, Kota Kinabalu, Malaysian Cocoa Board. AOAC. (1990). Offical methods of analysis. 15th edition, Washington DC, Association of Official Analytical Chemist. Biehl, B. and Meyer, B. (1990). Bean spreading: a method for pulp preconditioning to impair strong nib acidification during cocoa fermentation in Malaysia. J. Sci. Food Agric. 51: 35-45. Duncan, R.J.E., Godfrey, G., Yap, T.N., Pettipher, G.L. and Tharumarajah, T. (1989). Improvement of Malaysian cocoa bean flavour by modification of harvesting, fermentation and drying method – The Sime-Cadbury process. The Planter. 65: 157-173. Gur’eva, M.B. and Tserevitinov, O.B. (1979). Methods for evaluating the degree of fermentation of cocoa beans, USSR Patent No. 646254. Hii, C.L. (2002). Post harvest treatments for flavour quality enhancement in Malaysian cocoa beans. Proceeding of the MCTTAP Cocoa Technical Seminar, 2002, paper 5. Jinap, S. (1994). Organic acids in cocoa beans – A review. Asean Food Journal. 9(1): 3-12. Jinap, S., Thien, J. and Yap, T.N. (1994). Effect of drying on acidity and volatile fatty acids content of cocoa beans. J. Sci. Food Agric. 65: 67-75. Lopez, A.S. and Dimick, P.S. (1991). Enzymes involved in cacao curing. Food Enzymology. 2: 211-236. Meyer, B. and Biehl, B. (1989). Postharvest pod storage: a method for pulp preconditioning to impair strong nib acidification during cocoa fermentation in Malaysia. J Sci Food Agric. 48: 285-304. Misnawi, Jinap, S., Jamilah, B. and Nazamid, S. (2000). New approach in handling of under-fermented cocoa beans: reduction of polyphenols through enzymatic oxidation. Proceeding of the 7th ASEAN Food Conference, 2000, 342-348. Shahrir, S. and Mamot, S. (1987). Determination of fermentation Index (FI) and its application to cocoa quality and grading. Proceeding of FAMA-MCGC workshop of cocoa quality and grading, 1987, 40- 49.

Malaysian Cocoa Journal 64

D-FRUCTOSE ADDITION DURING MALAYSIAN COCOA NIBS ROASTING

Suzannah S. Cocoa Downstream Research Center, Malaysian Cocoa Board, Lot 3, Jalan P/9B Seksyen 13, 43650 Bandar Baru Bangi, Selangor Darul Ehsan, Malaysia

Malaysian Cocoa J. 1: 59-66 (2004) ABSTRACT Malaysian cocoa nibs were used to determine the effect of fructose addition and roasting temperature on the pH, moisture content, color, and flavor after roasting. The amount of fructose added exerted more effect compared to roasting temperature. The pH of roasted cocoa nibs decreases significantly with higher concentrations of fructose. Moisture content of roasted cocoa nibs was not significantly affected by the amount of fructose and roasting temperature. The cocoa liquor color (L and ‘a” values) significantly changed with different amount of fructose. All flavor attributes remain unchanged.

Key words : D-fructose, Malaysian cocoa nib, roasting

INTRODUCTION

Cocoa beans produced in Malaysia are characterised by excessive acidity, weak chocolate flavour, and the presence of undesirable flavours (Baigrie and Rumbelow, 1987). Malaysian beans were characterised as low pH (4.75 - 5.19) together with Brazil, East Cameroon, Indonesia, and Dominican Republic. Chocolates produced with beans from these regions also had a high percentage of bitter, and burnt flavour, a high percentage of weak chocolate flavour, and a low percentage of strong chocolate flavour (Jinap et al., 1995).

Cocoa flavours are produced mainly through the Maillard reaction during high temperature processing (roasting). The maillard reaction is greatly affected by the nature of the reacting amino acids or proteins as well as by the carbohydrate, the moisture content, pH, temperature, oxygen, metals, phosphates, sulfur dioxide and other inhibitors (de Man, 1990). The precursors of this reaction are amino acids, peptides, reducing sugars, and polyphenols (Rohan, 1963; Rohan and Stewart, 1966). During roasting the degradation of amino acids is incomplete whereas reducing sugar are completely consumed. The formation of reducing sugars such as glucose and fructose occurs during fermentation from sucrose hydrolysis. The concentration of both free amino acids and reducing sugars reach maximal values at about the same time and well within the duration of the normal fermentation period.

The objective of the present study is to evaluate the effect of fructose addition prior to roasting and roasting temperature on the characteristics of Malaysian cocoa liquor.

MATERIALS AND METHODS

Experimental Design - The study used Central Composite Design with two parameters; D-fructose concentration and roasting temperature at five different levels i.e. - α, -1, 0, +1 and + α. Table 1 shows the roasting experiment carried out with the amount of D-fructose added and roasting temperature used.

Roasting Trials - Cocoa nibs were prepared at the Cocoa Downstream Research Center, Bangi with cocoa beans obtained from Sg. Ruan, Pahang. Roasting experiments were carried out in a 15 kg capacity drum roaster (Barth, Germany). Eleven roasting experiments were conducted, with amounts of fructose added ranging from 0 to 2.5% and the roasting temperature from 130oC to 150oC. Each roasting used 15 kg of cocoa nibs and 1,500 ml of water into which sugars was dissolved. Sugar solutions were injected at the onset of the roasting process when the product temperature reached 70oC. The temperature was held at 70oC for 30 min, increased to the desired roasting temperature, and held at this temperature for 10 min. At the end of the roasting process the cocoa nibs were cooled immediately by blowing air to stop the roasting

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process. During roasting, the air temperature, product temperature, and drum pressure were monitored. At the end of roasting, samples were collected to determine pH and moisture content. Roasted cocoa nibs were then ground in a mortar and pestle mill to obtain cocoa liquor.

Table 1. Central Composite Design with α = 1.414

Test. No D-Fructose Roasting temperature Code % G Code Temperature (oC) PPF1 -1 0.36 54 -1 135 PPF2 1 2.14 321 -1 135 PPF3 -1 0.36 54 1 145 PPF4 1 2.14 321 1 145 PPF5 - α 0 0 0 140 PPF6 + α 2.5 375 0 140 PPF7 0 1.25 187.5 - α 130 PPF8 0 1.25 187.5 + α 150 PPF9 0 1.25 187.5 0 140 PPF10 0 1.25 187.5 0 140 PPF11 0 1.25 187.5 0 140

pH Determination - Ten (10) g of ground cocoa nibs were mixed with 90 ml boiling water. The solution was stirred using a glass rod for one min and filtered through Whatman # 4 filter paper. The pH was determined using a pH meter (Mettler Delta 320, Mettler-Toledo) equipped with temperature compensator. Triplicate determinations were made for each sample.

Moisture Content Determination - Moisture content was carried out using a halogen moisture analyzer (Mettler-Toledo, Switzerland). About 5 g ground cocoa nibs were used and two determinations were carried out for each sample.

Cocoa Liquor Preparation - Roasted cocoa nibs were ground in a mortar and pestle mill (Pascal Engineering) for about 4 hours to obtain a satisfactory particle size.

Cocoa Butter Preparation - Cocoa liquor was filtered using Whatman No. 4 filter paper in a convection oven (Memmert) set at 60oC. The cocoa liquor was left overnight to collect sufficient cocoa butter.

Color Determination - Color of the cocoa liquor were determined using a color meter (Hunter Colorlab). The instrument was standardized using a white tile, and the L, a, and b of the cocoa liquor was measured. The cocoa liquor was placed in a petri dish for measurement.

Sensory Evaluation - Nine-trained panelist consisting of the Cocoa Downstream Research Centre personnel evaluated the cocoa liquor for eight attributes: cocoa, bitter, astringent, acid/sour, fruity, moldy/earthy, raw/green, and viscosity. Cocoa liquors were scored based on Ghana cocoa liquor as a reference. Cocoa liquors were kept warm before being presented to panelist. Each session had three samples and one reference. Panelists were provided with water and plain biscuits to rinse their palette between samples.

Statistical Analysis - Data were analyzed using the SAS version 8.02 package (SAS Institute, Cary Indiana). The General Linear Model method was used to carry out analysis of variances.

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RESULTS AND DISCUSSION

Statistical analysis shows that pH was significantly (p<0.01) affected by the amount of D-fructose added but was not affected by the roasting temperature (Table 2).

Table 2. The estimated regression coefficient of roasted cocoa nibs pH

Parameter Estimate Standard T value Pr>t  error Intercept 4.9900 0.0174 286.23 <0.0001 Fructose -0.0716 0.0106 -6.71 0.0011** Roasting temperature 0.0183 0.0106 1.72 0.1468ns Fructose x Fructose 0.0206 0.0127 1.62 0.1655ns Roasting temperature x roasting 0.0056 0.0127 0.44 0.6767ns temperature Fructose x roasting temperature -0.028 0.0151 -1.82 0.1282ns

Figure 1 shows that pH decreases when the amount of fructose added is more and the roasting temperature is higher. Higher roasting temperature requires longer processing/roasting duration. Volatile acids may evaporate and raise the pH of the roasted cocoa nibs.

ph

5.2

5.1

5.0

2

EC HIP 1 e 50 s 1 1 to 45 c u 1 fr tem 40 p 1 35 1 0 30

Figure 1. The effect of fructose added and roasting temperature on pH of roasted cocoa nibs

Moisture content after roasting was not affected significantly by the amount of D-fructose added and roasting temperature (Table 3). At the end of the roasting process the moisture content of the cocoa nibs ranged from 0.95% to 1.75%. This increases slightly after the cocoa nibs had been cooled to room temperature (1.22% to 1.81%).

Table 4, 5 and 6 show the estimated regression coefficients for color analysis of cocoa liquor obtained from roasted cocoa nibs. For color analysis, the instrument used describes color in three different values: L, a and b. L describes the lightness or darkness ranging from 0 (black) to 100 (white), “a” ranges from –50 (green) to +50 (red) and “b” ranges from –50 (blue) to +50 (yellow).

The L value (brightness) and “a” value (red) were significantly affected by the amount of D- fructose added neither value was affected by the roasting temperature. The “b” value (yellow) was not significantly affected by either factor, i.e. amount of D-fructose added and the roasting temperature. Figure 2 and 3 illustrate the effect of fructose and roasting temperature on L value and “a” value respectively. The L value showed no large variation within the range tested, but the cocoa liquor became slightly darker at

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higher temperature. The cocoa liquor become redder (“a” value increases) at lower fructose content and higher roasting temperature.

Table 3. The estimated regression coefficient of roasted cocoa nibs moisture content

Parameter Estimate Standard T value Pr>t  error Intercept 1.6433 0.1202 13.67 <0.0001 Fructose 0.0719 0.0736 0.98 0.3733ns Roasting temperature 0.0379 0.0736 0.52 0.6282ns Fructose x Fructose 0.0114 0.0876 0.13 0.9011ns Roasting temperature x roasting -0.0711 0.0876 -0.81 0.4543ns temperature Fructose x roasting temperature 0.1050 0.1041 1.01 0.3595ns

Table 4. Estimated regression coefficient for L value of cocoa liquor Parameter Estimate Standard T value Pr>t  error Intercept 9.2700 0.1607 57.69 <0.0001 Fructose -0.4788 0.0984 -4.87 0.0046** Roasting temperature 0.02480 0.0984 0.25 0.8111 Fructose x Fructose -0.10376 0.1171 -0.89 0.4163 Roasting temperature x roasting -0.21880 0.1171 -0.187 0.1208 temperature Fructose x roasting temperature -0.1100 0.1392 -0.79 0.4651

Table 5. Estimated coefficient regression for a value of cocoa liquor

Parameter Estimate Standard T value Pr>t  error Intercept 5.8900 0.0733 80.26 <0.0001 Fructose -0.1214 0.0449 -2.70 0.0427* Roasting temperature 0.01660 0.0449 0.37 0.7270 Fructose x Fructose -0.0725 0.0535 -1.36 0.2333 Roasting temperature x roasting -0.0200 0.0535 -0.37 0.7238 temperature Fructose x roasting temperature -0.0175 0.0636 -0.28 0.7941

Table 6. Estimated coefficient regression for b value of cocoa liquor

Parameter Estimate Standard T value Pr>t  error Intercept 4.6133 0.1245 37.05 <0.0001 Fructose -0.1887 0.0762 -2.47 0.0562 Roasting temperature -0.0466 0.0762 -0.61 0.5675 Fructose x Fructose -0.0079 0.0908 -0.09 0.9340 Roasting temperature x roasting -0.0904 0.0908 -1.00 0.3649 temperature Fructose x roasting temperature -0.0875 0.1078 -0.81 0.4540

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L value

9.5

9.0

8.5

8.0 2

EC HIP 1 e 50 s 1 to 145 c u 1 fr tem 40 p 1 35 1 0 30

Figure 2. The effect of fructose added and roasting temperature on L value a value

5.9

5.8

5.7

5.6 2

EC HIP 1 e 50 s 1 1 to 45 c u 1 fr tem 40 p 1 35 1 0 30

Figure 3. The effect of fructose added and roasting temperature on a value

Statistical analysis indicates no significant difference in any of the sensory attributes analyzed, i.e cocoa, bitter, astringent, acid/sour, fruity, moldy/earthy, raw/green, and viscosity (Table 7).

Figure 4, 5, and 6 show the effect of fructose and roasting temperature on cocoa, astringent and acid attributes of cocoa liquor. The cocoa attribute shows an increase with higher content of fructose and lower roasting temperature. The amount of fructose added was limited to 2.5% because higher concentrations resulted in sticky cocoa nibs during roasting. This led to sticking of the cocoa nibs to the roaster drum and nibs eventually became burnt. Astringency increases at higher fructose concentration and lower roasting temperature. The acid attribute increases with higher fructose concentration and higher roasting temperature. This relates to the pH, which decreases at high fructose content.

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Cocoa

5.0

4.5

2

EC HIP 1 e 50 s 1 1 to 45 c ru te 140 f mp 135 1 0 30

Figure 4. The effect of fructose added and roasting temperature on cocoa attribute of cocoa liquor.

Astringent

3.8

3.6

3.4

2

3.2 EC HIP 1 e 50 s 1 1 to 45 c u 1 fr tem 40 p 1 35 1 0 30

Figure 5. The effect of fructose added and roasting temperature on astringent attribute of cocoa liquor

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Acid

3.4

3.2

3.0

2.8 2 EC HIP 1 e 50 s 1 1 to 45 c 1 fru tem 40 p 1 35 1 0 30

Figure 6. The effect of fructose added and roasting temperature on acid attributes of cocoa liquor.

CONCLUSION

The experimental conditions applied for the additions of D-fructose prior to cocoa nibs roasting significantly affect the pH of cocoa nibs and also the L value and ‘a’ value of cocoa liquor. However, the ‘b’ value and all the flavors attributes were not affected. One of the most important point to be observed in the improvement of cocoa flavor development through treatment such as fructose solution addition, is to ensure intensive contact between the reacting components – e.g fructose and amino acids. Boller and Braun (2000) suggest that cocoa mass may be a better vehicle for this type of treatment compared to cocoa nibs due to the microstructure of the cocoa particles present in the cocoa mass.

ACKNOWLEDGEMENT

Research supported by Intensive Research in Priority Area (IRPA) of Ministry of Sciences, Technology and Innovation, Malaysia (03-04-07-0108).

REFERENCES

Baigrie, B.D. and Rumbelow, S.J. (1987). Investigation of flavor defects in Asian cocoa liquors. J. Sci. Food Agric., 39: 357-368 Boller, E. and Braun, P. (2000). Optimal Flavor Development. Chocolate Production Technology-Part 1. Buhler AG CH-9240 Uzwil de Man, J.M., (1990). Principles of Food Chemistry. 2nd Ed. Van Nostrand Reinhold, 115 Fifth Avenue, New York, NY . pp. 100-112 Jinap, S., Dimick, P.S., and Hollender, R. (1995). Flavor evaluation of chocolate formulated from cocoa beans from different countries. Food Control, 6: 105-110 Kattenberg, H.R. and Kemmink, A., (1993). The flavor of cocoa in relation to the origin and processing of the cocoa beans. Food flavours, Ingredients and composition. Charalambous, G. (Ed.). p. 1-2 Rohan, T.A. (1963). Precursors of chocolate aroma. J. Sci. Food Agric., 14: 799-805 Rohan, T.A. and Stewart, (1966). The precursors of chocolate aroma: Changes in the free amino acids during roasting of cocoa beans. J. Food Sci. 31: 202-205

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Table 7. Pr value for sensory attributes of cocoa liquor

Parameter Cocoa Bitter Astringent Acid Fruity Raw Smoky Moldy Viscous Intercept <0.0.001 <0.0001 <0.0001 <0.0001 0.1919 0.0005 0.9998 0.0250 <0.0001 Fructose 0.2177 0.5216 0.2204 0.1298 0.9276 1.0000 0.2531 1.0000 0.7210

Roasting 0.9446 0.4226 0.4156 0.4980 0.6905 1.0000 0.2531 1.0000 0.4275 temperature Fructose x 0.1208 0.4133 0.6287 0.5826 0.3220 0.0029 0.6107 0.0822 0.4168 Fructose Roasting 0.2588 0.1387 0.1833 0.1383 0.9507 0.0823 0.6107 0.3275 0.1195 temperature x roasting temperature Fructose x 0.5123 0.1089 0.6416 0.5965 0.7573 0.0409 0.1274 0.0409 0.2868 roasting temperature

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EFFECTS OF BLENDING COCOA BUTTER AND CAROTINO OIL ON THE PHYSICAL AND SENSORY QUALITIES OF FAT BLENDS AND CHOCOLATE

Wan Aidah W.I. 1, H. Asimah1, B. Abd. Salam2 and S. Mamot2 1 Cocoa Downstream Research Center, Malaysian Cocoa Board, Lot 3, Jalan P/9B Seksyen 13, 43650 Bandar Baru Bangi, Selangor Darul Ehsan, Malaysia 2 Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

Malaysian Cocoa J. 1: 67-73 (2004) ABSTRACT Carotino oil contains valuable nutrients and is a rich source of natural carotenoids, vitamin E, and co-enzyme Q10. This oil can be added to chocolate in order to boost the nutrients in chocolate. However, the blending of carotino oil and cocoa butter may change the physical quality of chocolate and therefore its eating characteristics. In this study, cocoa butter (CB) and carotino oil (CO) were blended in four different weight ratios: 100:0, 90:10, 80:20 and 70:30. Fat blends were then used to produce milk chocolate. The fat blends and the chocolate samples were then tested for melting profile using a differential scanning calorimeter (DSC) and solid fat content using a nuclear magnetic resonance spectrometer (NMR). The DSC test showed that the hardness of the fat blends and chocolate decreased with the addition of carotino oil. This is supported by sensory evaluation. However both sensory and DSC analyses showed that the addition of 10% carotino oil in the fat system did not result in a significant change to the hardness of both the fat blend or chocolate. NMR analysis showed that pure cocoa butter and the different ratios of the CB:CO blends followed the same SFC profile. However, the SFC decreased with increasing in the addition of carotino oil, and the cooling rate also decreased with increasing CO. Sensory evaluation showed that chocolate with higher carotino oil content melted rapidly in the mouth. This is supported by SFC measurements where the blend with higher carotino oil required a lower temperature for the fat crystals to fully melt. However with 10% carotino oil in the fat blend, no significant changes were observed in terms of melting behavior as shown both by sensory evaluation and SFC for chocolate. Sensory evaluation also showed that the panels like all the products equally regardless of the amount of carotino oil used.

Key words: Carotino oil, Chocolate, Cocoa butter, Melting profile, Solid fat content

INTRODUCTION

Carotino oil is a refined vegetable oil rich in natural carotenoids, vitamin E and co-enzyme Q10 (Kritchevsky, 2000). Commercial carotino red palm oil contains not less than 800 ppm vitamin E and approximately 500 ppm carotene, 90% of which present in the form of α- and β-carotene (Nagendran et al., 2000). Beta-carotene is a precursor for vitamin A and also an effective antioxidant. A diet rich in β-carotene, vitamin E and other antioxidants is believed to exhibit health benefits by helping to prevent certain diseases such as cancer, coronary heart diseases, and immune malfunctions (Bendich, 1990; Mascio et al., 1991; Weisburger, 1991). Studies have also shown that carotino red palm oil can be used as a vitamin A fortification and can be applied in the baking industry (Benede, 2001; Stuijvenberg et al., 1999). The co-enzyme Q10 is a newly discovered food component known to give some benefits to health. It has a potential to boost immune system, relieve angina, protect against heart disease, and reduce high blood pressure (Goh, 1996).

Carotino oil contains valuable nutrients and has been widely used by health conscious people in their daily food preparation such as for frying, salad dressing and recently for baking. Another potential area where the benefit of carotino oil can be applied is in the production of chocolate. Chocolate is eaten all over the world and is well enjoyed by many, quite simply because people find pleasure in it. However some people associate chocolate with negatives effect such as obesity, tooth decay and heart disease. Even though many misconceptions about chocolate have been scientifically proven wrong (Grenby, 1974; Waterhouse et al., 1996; Anon., 1999), it is difficult to change people’s perception towards chocolate. Adding a positive value in chocolate such as in this case chocolate rich in antioxidants (carotene and vitamin E from carotino oil), may help to boost the perception on the goodness of chocolate and promote its consumption.

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Chocolate has two major distinguishing characteristics, its flavor and texture. Although many different flavors of chocolate exist, all must be free from objectionable taste, and yet incorporate at least some of the pleasant ones, which the consumer will associate with the product. A primary feature of the texture is that it must be solid at room temperature and yet melt rapidly in the mouth at 37°C, giving a liquid that appears smooth to the tongue (Beckett, 1994). The fat in chocolate determines its eating characteristics such as melting behavior, flavor release, heat resistance and consistency (Anon., 1994). The blending of carotino oil with cocoa butter may change the physical quality of chocolate and thus alters its eating properties. Therefore the purpose of this study is to discover the effect of blending cocoa butter with carotino oil on the physical and sensory quality of the fat blend and the chocolate.

MATERIALS AND METHODS

Materials - Pure prime pressed cocoa butter was obtained from KL-Kepong Cocoa Products Sdn. Bhd., Malaysia and Carotino oil (the product from Carotino Sdn.Bhd., Malaysia) was purchased from a local market.

Fat Blend Preparation - Cocoa butter was melted at 50°C in an oven prior to use. Liquid carotino oil (CO) was blended with the melted cocoa butter (CB) by a mixture design in proportions of CO ranging from 0 to 50%. The fat blends were then cooled to set in a chiller at 15°C before being left at room temperature overnight. The blends, that stayed solid at room temperature were used as samples for this study. Four blends were chosen: 100:0, 90:10, 80:20 and 70:30, identified by weight ratio of CB:CO.

Chocolate Production - The four fat blend samples were used in the making of four milk chocolate samples. The formulation of the milk chocolate was based on Beckett (1994). Cocoa liquor was mixed with refined sugar, milk powder and part of the fat blend in a lab scale mortar and pestle mill (Pascal, U.K), with 1 kg capacity for 10 minute at 45°C to form a paste. The paste was then refined using a three roll refiner (Pascal, U.K) to get a particle size of less than 35µm measured by a micro screw meter (Mitutoyo, Japan). The mass was then transferred back into the mortar and pestle mill with the remaining portion of the fat blend for a conching process of 16 hours at 55°C. Two hours before the conching ended, lecithin and vanillin was added. The chocolate was tempered manually on a marble slab at 28°C to 29°C and then moulded into a bar shape.

Melting Profile - Melting profile was measured using a differential scanning calorimeter DSC-7 (Perkin Elmer, Norwalk, Connecticut, USA). The method used was based on Md. Ali & Dimick (1994). The fat sample was melted in an oven at 50°C. A sample weighing 3-5mg was hermetically sealed in an aluminum pan. The sample were heated to 60°C for 30 min and then cooled at 0°C for 90 min. It was then transferred into an incubator at 26°C for 40 hours for stabilization. The sample was cooled again at 0°C and held for 90 min before it was transferred to a DSC chamber and held at -25°C for 5 min on the DSC head. The melting profile of the fat was measured at a heating rate of 20°C/min from -25°C to a maximum of 50°C.

Solid Fat Content (SFC) - Solid fat content was measured using a Nuclear Magnetic Resonance spectrometer, Newport Analyzer Mark 3 (Newport Pagnell, England). The method used was as described by Nilsson (1986). The sample in the NMR tube was first melted at 80°C and then held at 60°C for 20 min. It was then cooled at 0°C for 90 min. The sample was then kept at 26.5°C for 40 hours followed by cooling at 0°C for 90 min. The sample was then stabilized for 35 min at each measuring temperature of 10°C, 20°C, 25°C, 27.5°C, 30°C, 32.5°C, 35°C and 40°C prior to the measurement of SFC.

Sensory Evaluation - Sensory evaluation of chocolate was conducted by 20 experienced in-house panelists. The method used was a descriptive analysis with scaling (Larmond, 1982) of 1 to 7 where 1 is the lowest intensity and 7 is the highest. The panelists were asked to evaluate only the three sensory parameters that are related to the effect of the fat composition in the samples. The parameters tested were hardness, which was defined as the hardness felt during the first bite of chocolate, from very soft to very hard; meltdown, which was defined as the amount of melted chocolate after 30s in the mouth, from very little to very much melted; and lastly the overall acceptance, which describe how much the panel likes the sample, from dislike very much to like very much.

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Statistical Analysis - The data was analyzed for analysis of variance (ANOVA) and Duncan Multiple Comparison Test at a significance level of 0.05 (SPSS 10.0, USA).

RESULTS AND DISCUSSION Melting profile

Figure 1 shows the melting thermograms of different blends of cocoa butter and carotino oils. The onset, the peak and the end (offset) temperatures of the fat blends are shown in Table 1. The onset temperatures represent the starting temperature where the fat crystals begin to melt, which involves the melting of the low melting point triglycerides, whereas the end or the offset temperature indicates the end of the melting process (Breitshuh and Windhab, 1996). A shift from the solid phase to liquid phase occurred at the peak temperature (Yap et al., 1989). The maximum energy is absorbed at this point, where it involves the melting of the high melting point fat crystals. Our results show that these temperatures decrease as the proportion of carotino oil increases. Taking the peak value as the melting temperature, it shows a decrease from 35.07°C with pure cocoa butter and decreasing with the addition of carotino oil to 32.74°C with 30% carotino oil. However as shown in Table 1, the differences are not significant (at p=0.05), except with the pure cocoa butter.

The melting of fat crystals involves the absorption of energy (endothermic), which is calculated through the enthalpy value (∆H). This is defined as the amount of energy needed to melt all the fat crystals. As shown in the results the amount of ∆H decreases significantly with the addition of carotino oil to cocoa butter, from 124.6J/g (without CO) to 89.4J/g (with 30% CO). According to Sabariah et al. (1998) the enthalpy value is proportionally related to the hardness of the fat. Therefore the higher the carotino oil added to the system, the softer is the fat blend.

A B

C Endothermic D

-25 -20 -10 0 10 20 30 40 50

Temperature (°C)

Figure 1. DSC melting thermograms of a blend of cocoa butter and carotino oil at different weight ratios: (A)100:0, (B)90:10, (C)80:20 and (D)70:30.

Table 1. Mean value for onset (°C), End (°C), Peak (°C) and ∆H (J/g) for blends of cocoa butter and carotino oil as obtained by DSC.

Sample Onset (°C) End (°C) Peak (°C) ∆H (J/g) CB:CO (w/w) 100:0 28.489b 39.676b 35.070b 124.575d 90:10 27.257a 37.229a 33.402a 114.308c 80:20 27.507a 36.824a 33.070a 104.174b 70:30 27.415a 36.125a 32.735a 89.355a Mean value followed by the same alphabet in the same column are not significantly different at p≥0.05

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Table 2 and Figure 2 show the results obtained for the melting profile of chocolate made from different ratios of cocoa butter and carotino oil. The onset, the end and the peak temperatures are not significantly different (at p=0.05) for the chocolate made from different proportions of CB:CO. The results obtained correlate to that of the pure fat blend. The softening effect of the fat blend was also observed in chocolates with increasing amounts of carotino oils added.

Table 2. Mean value for onset (°C), End (°C), Peak (°C) and ∆H (J/g) for chocolate with carotino oil as obtained by DSC

Sample Onset (°C) End (°C) Peak (°C) ∆ H (J/g) CB:CO (w/w) 100:0 29.549 a 37.117 a 34.737 a 24.933ab 90:10 29.224 a 37.314 a 34.402 a 27.002b 80:20 30.069 a 37.863 a 35.066 a 25.955ab 70:30 28.617 a 37.360 a 34.735 a 18.518a Mean value followed by the same alphabet in the same column are not significantly different at p≥0.05

A

c c

i i

m m r

r B

e e

h h

t t

o o

d d n

n C

E E D

-25 -20-10 010203040 Temperature (°C) Figure 2. DSC melting thermograms of chocolate with different blends of CB:CO : (A)100:0, (B)90:10, (C)80:20 and (D)70:30 by weight ratio.

Solid Fat Content (SFC)

The characteristics of fat can be visualized by measuring the percentage of solid fat content at various temperatures. Figure 3 shows the SFC profiles of different ratios of CB:CO blends. Pure cocoa butter (100:0 blend) has a unique melting profile, where the hard fat becomes liquid after a comparatively small rise in temperature. This is shown by a sharp drop in SFC from 27°C to 33°C. This quick meltdown of the fat results in a cooling sensation in the mouth. As observed, different ratios of CB:CO blends had similar SFC profiles and their SFC decreases with increasing carotino oil. All blends gave a sharp drop in SFC at temperature from 27°C to 33°C, which will give a cool feeling in the mouth while the flavor is released. However with higher carotino oil content, the sharpness of the drop decreases and results in lower cooling effect to the mouth. The higher the carotino oil content in the fat blend, the lower the temperature needed to melt all the fat crystals. At 35°C all the fat solids are melted which shows that all the fat blends did not give a waxy mouthfeel.

The melting profile as measured by the solid fat content with temperature is one of the most important properties of chocolate because it determines its eating characteristics such as melting behavior, flavor release, heat resistance and consistency (Anon., 1994). Figure 4 shows the SFC profiles for the chocolate made from different ratio of CB:CO blends. As observed the chocolate with different ratios of CB:CO had similar SFC profiles and their SFC decreased with increasing carotino

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oil. It showed a similar trend to that of pure fat blend. However chocolate with 10% carotino oil did not differ significantly (at p=0.05) in the melting profile as compared to the chocolate with pure cocoa butter. This shows that chocolate with 10% carotino oil will have almost similar eating characteristics as the pure chocolate.

Molten chocolate is a suspension of particles of sugar, cocoa and milk solids in a continuous fat phase (Beckett, 1994). Because of the presence of solid particles in the melted state, the SFC reading in chocolate did not go down to zero (Figure 4).

90 80

80 75 70 70 60 SFC 65 SFC (%) 50 100:0 (%) 090:10 60 40 80:20 100:0 70:30 90:10 30 55 80:20 70:30 20 50 10 45 0 10 15 20 25 30 35 40 45 40 10 20 30 40 50 Temperature (°C) Temperature (°C) Figure 3. Solid fat content of Figure 4. Solid fat content of different ratios of CB:CO chocolate made from different (w/w). ratios of CB:CO (w/w)

Sensory Evaluation

Figure 5 shows the mean flavor scores of chocolate made from different ratios of CB:CO blends as presented in a web-diagram. Results show that chocolate with 10% carotino oils does not differ significantly (at p=0.05) in terms of hardness and meltdown with chocolate made from 100% cocoa butter. This sensory result correlates with the result obtained by SFC analysis. The hardness of chocolate however decreases significantly with the increase in carotino oil. The result also correlates with the value of ∆H as obtained by DSC analysis. The meltdown or the rate that chocolate melts in the mouth increases with the addition of carotino oil where chocolate with 30% carotino oil melts the fastest. This result also correlates with the SFC result. However, the preference or acceptability of the chocolates made from different blend ratios of CB:CO showed no significant difference. The panels liked the products equally regardless of the amount of carotino oil added.

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hardness* 6

4 100:00 90:10 80:20 2 70:30

0

acceptability meltdown*

*significantly different at p=0.05

Figure 5: Flavor profile of chocolate with different ratio of CB:CO (w/w).

CONCLUSIONS

The blending of carotino oil and cocoa butter will change the physical properties of chocolate and thus alters its eating properties. The DSC study showed that a significant reduction in enthalpy value, which is the energy needed to melt the crystals, was observed with the addition of carotino oil. This is proportionally related to the hardness of the fat system, where the more carotino oil is added, the softer the fat. Similar results were obtained for the chocolate. This result correlates with the sensory evaluation result, which shows that the hardness of chocolate decreases significantly with the addition of carotino oil. However both the sensory and DSC analyses show that 10% carotino oil in the fat system did not significantly change (at p=0.05) the hardness of both the fat blend and the chocolate.

The solid fat content measurement shows that the pure cocoa butter and the different ratios of CB:CO blends followed the same melting profile. The sharp drop of SFC between 27°C to 33°C indicates that the fat system exhibits the cooling effect to the mouth, which is required during flavor release in chocolate. However, this effect decreases with an increase in the addition of carotino oil as shown by the decrease in the tangent of the SFC curve between 27°C and 33°C.

Chocolate with higher carotino oil content melts more rapidly in the mouth. This is shown in the sensory evaluation result and is supported by the melting profile result from the SFC measurement, where blends with higher carotino oil required a lower temperature for the fat crystals to fully melt. However with 10% carotino oil in the fat blend, no significant changes were observed in the melting behavior. This is as shown in both the sensory result and the SFC result for chocolate. The sensory evaluation result also shows that the panels like all the products equally regardless of the amount of carotino oil used in the study. Therefore to fully utilize its benefit, a maximum amount of 30% carotino oil is recommended for the fat blends for the production of chocolate. The use of 10% carotino oil in the fat blend will maintain the physical and sensory qualities similar to chocolate made from pure cocoa butter.

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ACKNOWLEDGEMENTS

The authors acknowledge the technical support received from the staff of the Chemistry and Technology Division, Malaysian Cocoa Board and the Faculty of Science and Technology, Universiti Kebangsaan Malaysia.

REFERENCES

Anon. (1994). Facts about fats, Loders Crooklaan,Wormerveer, Holland. Anon. (1999). Nutritional functions of cocoa and chocolate in human food. ADM Cocoa B.V. DeZaan. Benede, A.J.S. (2001). The potential of red palm oil based shortening as food fortification for vitamin A in the baking industry. Food Nutr. Bull. 22, 416-418. Bendich, A. (1990). Antioxidant nutrients and immune functions. Advances in experimental medicine and biology. New York, Plenum Press, pp. 75-112. Beckett, S.T. (1994). Industrial Chocolate Manufacture and use, 2nd edition. Chapman & Hall, London, pp. 139, 276. Breitshuh, B. and Windhab, E. (1996). Direct measurement of thermal fat crystal properties of milk fat fractionation. J. Am. Oil Chem. Soc. 73(11), 1603-1610. Goh, S.H. (1996). Coenzyme Q. Malaysian Oil. Sci Tech. 5, 19. Grenby, T.H. (1974). The deposition of dental plaque in young adults on a diet containing chocolate and skim milk powder. Arch. Oral Biol. 19, 213. Kritchevsky, D. (2000). Impact of red palm oil on human nutrition and health. Food Nutr. Bull. 21, 182-188. Larmond, E. (1982). Laboratory Method For Sensory Evaluation of Food. Canada Department of Agriculture, Ottawa, Canada, pp. 48-55. Mascio, P.D.; Murphy, M.E.; Sies, H. (1991). Antioxidant defense system: The role of carotenoids, tocopherols and thiols. Am. J. Clin. Nutr. 53, 226-237. Md.Ali, A.R. and Dimick, P.S. (1994). Thermal analysis of palm mid-fraction, cocoa butter and milk fat blends by differential scanning calorimetry. J. Am. Oil Chem. Soc. 71(3), 299-302. Nagendran, B.; Unithan, U.R. Choo, Y.M. and Sundram, K. (2000). Characteristic of red palm oil, a carotene- and vitamin E-rich refined oil for food uses. Food Nutr. Bull. 21, 189-194. Nilsson, J. (1986). Measuring solid fat content. 40th P.M.C.A. Production Conference, pp 81-84. Sabariah, S.; Md. Ali, A.R. and Chong, C.L. (1998). Physical properties of Malaysian cocoa butter as affected by addition of milk fat and cocoa butter equivalent. Int. Food Sci. Nutr. 49, 211-218. Stuijvenberg, M.E.; Kvalsvig, J.D.; Faber, M.; Kruger, M.; Kenoyer, D.G. and Benede, A.J.S. (1999). Effect of iron, iodine and Beta carotene-fortified biscuits on the micro nutrient status of primary school chidren: A randomized controlled trial. Am. J. Clin. Nutr. 69, 497-503. Waterhouse, A.L.; Shirley JR; Danovan J.L. (1996). Antioxidants in chocolate. The Lancet. 348, 834. Weisburger, J.H. (1991). Nutritional approach to cancer prevention with emphasis on vitamins, antioxidants and carotenoids. Am. J. Clin. Nutr. 53, 226-237. Yap, P.H.; deMan, J.M. and deMan, L. (1989). Polymorphism of palm oil and palm oil products. J. Am. Oil Chem. Soc. 66, 693-697.

Malaysian Cocoa Journal 79

DETERMINATION OF HYDRO DISTILLED ESSENTIAL OILS IN THEOBROMA CACAO L.

Samuel Y. K. C. 1, A. M. Sri Nurestri2 and R. Nazarudin2 1 Cocoa Downstream Research Center, Malaysian Cocoa Board, Lot 3, Jalan P/9B Seksyen 13, 43650 Bandar Baru Bangi, Selangor Darul Ehsan, Malaysia 2 Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

Malaysian Cocoa J. 1: 74-82 (2004) ABSTRACT Hydro distilled essential oils from the cocoa leaves, branches, flowers and pods of UIT 1 and NA 33 were investigated qualitatively by gas chromatography and gas chromatography-mass spectrometry (GC-MS). The main components of the oils were identified by comparison of their mass spectral data with those of the mass spectral reference library. Retention indices relative to n-alkane were determined. Study showed that hydro distilled essential oils from cocoa pods and flowers gave little flavor compounds. Fatty acid was the major constituents. Hydro distilled essential oils from leaves contained the most volatile compounds compared to other plant parts. Linalool oxide and linalool could be found in all the samples. Genetic differences, different geographical location and cropping season produces varying constituents of essential oils in either the concentration pattern or the lack of certain volatiles.

Key words: Cocoa, Hydro distilled essential oils, Shikimic acid phenylpropanoid pathway, Linalool.

INTRODUCTION

Volatile oils are the odorous principles found in various plant parts. When they are exposed to air at ordinary temperatures, they evaporated; therefore they are called volatile oils, ethereal oils, or essential oils. The last term is applied because the oils represent the “essences” or odor constituents of the plants.

Essential oils are highly concentrated plant extract. It is highly volatile with characteristic odors. Many are non-oily (more like water than oil); though most are oily feeling. It is usually lighter than water and insoluble in water. However, it will impart odor to water. Essential oils are soluble in alcohol, ether, fixed oils, and organic solvents. Although differing in their chemical constitution, essential oils have many physical properties in common such as boiling points vary from 160 to 240°C, densities are from 0.759 to 1.096, high refractive indices, most are optically active, specific rotation is often a valuable diagnostic property mixtures of many constituents (Bauer and Garbe, 1985).

Chemically, essential oils are extremely complex mixtures containing compounds of every major functional group class (Parker, 1993). Monoterpene hydrocarbons make up the bulk of many essential oils (Heywood, 1982). Essential oils are valuable natural products used as raw materials in many fields, including perfume, cosmetics, aromatheraphy and phytotheraphy, spices and nutrition (Buchbauer, 2000).

The objective of this paper is to report the chemical compounds of the essential oils in cocoa with reference to genotype, various plant parts, season and geographical locations.

MATERIALS AND METHODS

Experiment Design - An experiment consisting of two different genotypes of cocoa (UIT 1 and NA 33); four different parts of the plant (leaves, branches, pods and flowers), two locations (Hilir Perak and Tawau) and two cropping seasons was carried out.

Sample Collection and Preparation - The fresh sample was collected and sun-dried. Samples were ground by using the Waring blender.100 g of the samples were weighted and transferred to the 2L

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flask. The flask was filled with water until 2/3 full and some anti bumping granular were added. The flask was heated by heating mantel until boiling and distilled for 6 hours (Zubrick, 1984).

Essential Oil Extraction - Sodium chloride (10% of the distillate) was added and dissolved into the distillate. The distillate was transferred into a big separating funnel (1 L). The receiver flask was washed with 25 ml dichloromethane. The distillate was extracted by twice with 25 ml dichloromethane. The pool of organic layer was dried using sodium sulfate anhydrous and filtered. Dichloromethane was evaporated using rotorvapor at 40°C (Zubrick, 1984; Charles, 1988).

Chemical Compositions Determination - Chemical compositions of the essential oils obtained were determined using GC/GCMS:

Instrument HP 6890 gas chromatograph (Hewlett Packard), USA for GCMS Shimadzu GC-17A, Japan for GC/FID Column HP INNOWax (polar) Column phase: polyethylene glycol (30m x 0.32 mm x 0.25 µm) Column temperature From 60°C to 230°C at 4°C/min, follow by 7.50 min. Isothermal at 230°C Carrier gas Helium Injection volume 1.0µL Injection mode Splitless MS detector Mass selective detector running in 70eV at 10-5 Torr Peak intergration ChemStation Autointergrator Analysis Identified by comparison with published mass spectra (Anon., 1990; Anon., 2000). Retention indices were determined relative to n-alkanes in accordance with established method (Harris, 1982; Sun et al., 1993).

Refractive index was determined using ATAGO model R-5000 Hand Refractometer at (26 ± 1)°C.

RESULTS AND DISCUSSION

It was noticed that hydro distilled essential oils from all plant parts were lighter than water and yellowish in color. Study showed that hydro distilled essential oils from cocoa pod gave little flavor compounds of interested but fatty acid in bulk (Table 1). Hence, further study on pod was discarded.

Table 1 Composition of hydro distilled cocoa pod oils

No.. Chemical compositions Area peak (%) 1 1-hexanol 1.76 2 Linalool oxide 1.57 3 Propanoic acid 3.88 4 Hexanoic acid 11.15 5 Dodecanoic acid 16.31 6 Tetradecanoic acid 8.73 7 Hexadecanoic acid 36.20

Generally, not many flavoring compounds were found in hydro distilled cocoa flower oils for both geographical locations (Table 2). Fatty acid was the major constituents. This was supported by the fact that cocoa flower was almost odorless (Minifie, 1982). Existence of fatty acids were expected because glycerol esters of fatty acids make up to 99% of the lipids of plant, which together with proteins and carbohydrates, constitute the principal structural components of all living cells (Fennema, 1985).

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Table 2. Composition of hydro distilled cocoa flower oils from Tawau and Hilir Perak

Chemical name Tawau Hilir Perak (percentage area) (percentage area) Epoxylinalol 3.85 2.26 4-ethoxy-ethyl ester (C11H14O3) 4.08 2.57 Decanoic acid 1.67 - Dodecanoic acid 11.04 7.13 Tetradecanoic acid 7.60 8.03 Pentadecanoic acid 3.87 6.11 Hexadecanoic acid 2.41 2.96 Hexadecanoic acid 32.60 39.78 9-Hexadecenoid acid 10.67 14.35 14-pentadecenoic acid 3.42 - Linoleic acid 11.52 - 11,14,17-eicosatrienoic acid methyl ester 2.70 - 13-methyl-oxacyclotetradecane-2,11-dione - 2.83 Heptadecene-(8)-carbonic acid (C18H34O2) - 5.68 Cyclodecene - 8.31

Hydro distilled essential oils of branches, leaves, and flowers for all samples had a refractive index ranging from 1.373-1.384 at 25°C (Table 3). Physical test parameter of refractive index could be helpful in confirming or rejecting the authenticity of oils declared botanical species and country of origin, whilst possibly revealing any adulteration with a foreign substances. For instance, Anise oil has a refractive index of 1.553-1.558 at 20°C (www.pinechemicals.biz, 2002) whereas Blackcurrant Bud (Ribes nigrum) has a refractive index of 1.355-1.365 at 20°C.

Table 3. Refractive index at 25°C of hydro distilled essential oils of cocoa branches, leaves, and flowers

Clones Cropping season Location Branches Leaves Flower* Peak Tawau 1.383 1.382 1.374 UIT1 Hilir Perak 1.384 1.382 1.373 Off Tawau 1.381 - Hilir Perak 1.384 1.380 - Peak Tawau 1.379 1.380 - NA33 Hilir Perak 1.379 1.382 - Off Tawau 1.379 1.381 - Hilir Perak 1.384 1.379 - * Dropped cocoa flowers were collected not according to the clone.

Linalool and linalool oxide were detected in all the samples. However, (Z)-3-hexenol, α- terpineol and benzylalcohol could be found in most of the samples. Beside linalool, α-ionone, benzylalcohol, farnecyl acetone and phytol were the common compounds found in hydro distilled leaves oils. For hydro distilled branches oils, compounds such as linalool oxide, linalool, α-terpineol and epoxylinalol were detected for both clones from two different geographical locations and two different cropping seasons (Table 4).

Farnesyl acetone and β-ionone were the only compounds that could be detected in all the leaves samples but not in other plant parts. Other important major flavoring compounds not detect or not existing in branches samples include 2-hexenal, 6-methyl-5-heptadien-2-one, β-damascenone, (E)- nerolidol, farnesol and phytol. Most of the major volatiles existed in branches samples could be found in leaves samples except terpin-4-ol, benzenaacetaldehyde, and isoeugenol.

Compounds in essential oils obtained could be categorized into four categories; aliphatic compounds, terpenoid and its derivatives, benzene and its derivatives and nitrogen compounds (Oyen

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& Nguyen, 1999). Linalool oxide (furanoid form), linalool, terpinen-4-ol, α-terpineol, epoxylinalol, nerol, geraniol, nerolidol, farnesol, phytol, and 2-hydroxy-cyclopentadecanone are categorized as terpenoid and its derivatives. Epoxylinalol was also known as linalool oxide in pyranoid form.

Benzaldehyde, benzenaacetaldehyde, 2-hydroxy-benzaldehyde, methyl salicylate, 2-methoxy- phenol, benzylalcohol, 2,6-bis-(1,1-dimethylethyl)-4-methylphenol, benzeethanol, eugenol, 4-ethoxy- ethylester-benzoic acid, 4-ethoxy-benzoic acid, 2,4-bis-(1,1-dimethylethyl)phenol, 2-methoxy-4-(1- propenyl)phenol, 4-vinyl-2-methoxy-phenol, isoeugenol, and 3,4-dihydro-1H-2-benzopyran-1-one are categorized as benzene and its derivatives.

Indole is categorized as nitrogen compounds. Others were in category of aliphatic compounds. It was believed that terpenoid and its derivatives were produced by the terpenoid biosynthesis via acetic acid-mevalonic pathway. Phenolic and its derivatives compounds such as methyl salicylate, 2- methoxy-phenol, 2,6-bis-(1,1-dimethylethyl)-4-methylphenol, eugenol, 2,4-bis-(1,1- dimethylethyl)phenol, 2-methoxy-4-(1-propenyl)phenol, 4-vinyl-2-methoxy-phenol, isoeugenol, and 3,4-dihydro-1H-2-benzopyran-1-one were produced from shikimic acid phenylpropanoid pathway (Henning et al., 1959; Fujii et al., 1980; Bloch et al., 1959; Knee, 2001).

Table 5 showed that different clones from different geographical location and cropping season would result in different hydro distilled essential oils compositions and contents. Fatty acids could be found in all hydro distilled essential oils from cocoa plant parts and hexadecanoic acid appeared as dominant in most of the samples. Hydro distilled leaf oils from peak season had less fatty acids contents (4.49%-9.42%) if compared to those from off season.

The formation of essential oils in the plant, and consequently the yield and composition of the oil produced, depends on many factors. Genetic differences in plants of the same species that are otherwise indistinguishable can result in widely different essential oil content. Geographical location and agricultural factors also influence oil production (Heywood, 1982). Besides, the composition of an essential oil is dependent on production technique and purity. Therefore, the same plant from different geographic origin produces varying constituents of essential oil in either the concentration pattern or the lack of certain volatiles (Buchbauer, 2000).

CONCLUSION

It was noticed that hydro distilled essential oils from cocoa leaves and branches contained functional groups such as alcohol, aldehyde, ketones, etc. Terpene compounds could not be detected or does not exist. Generally, hydro distilled essential oils from cocoa leaves gave more compounds compared to other plant parts. Not many flavoring compounds could be detected from hydro distilled essential oils from dropped cocoa flowers and cocoa pod.

REFERENCE

Anon. (1990). Wiley 138K. Mass Spectral Database. John Wiley & sons. USA. Anon. (2000). Wiley 275K. Mass Spectral database. John Wiley & sons. USA. Bauer, K., and Garbe, D. (1985). Common Fragrance and Flavor Materials. Preparation, properties and uses. VCH, Germany. Bloch, K., Chaykin, S., Philips, A. H. and de Waard, A. (1959). Mevalonic acid phyrophosphate and isopentenylpyrophosphate. J. Biol. Chem. 234: 2595- 2604. Buchbauer, G. (2000). The Detailed Analysis of Essential Oils Leads To The Understanding of Their Properties. Perfumer & Flavorist., 25(2). p.64-67. Charles, F. Wilcox, Jr. (1988). Experimental Organic Chemistry. A small-scale approach. Macmillan Publishing Company. New York. Fennema, O.W. (1985). Food Chemistry second Edition, Revised and Expanded. Marcel Dekker INC. New York. Fujii, H., Segami, H., Koyama, T., Ogura, K., Seto, S., Baba, T. & Allen, C. M. (1980). Variable Product Specificity of Solanesyl pyrophosphate Synthetase. Biochem. Biophy. Res. Commun. 96: 1648-1653. Harris, D.C., (1982). Quantitative Chemical; Analysis. W.H. Freeman and Company, New York.

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Henning, U., Moslein, E. M. & Lynen, F. (1959). Biosynthesis of terpenes. Formation of 5- pyrophosphomevalonoc acid by phosphomevalonic kinase. Arch. Biochem. Biophys. 83: 259-267. Heywood, V.H. (1982). Popular Encyclopedia of Plants. Cambrige University Press. London. Knee, M. (2001). Terpenoids and Phenolics. USA: The Ohio State University. Minifie, B.W. (1982). Raw cacao-Botany and cultivation: Chocolate, Cocoa and Confectionery Science and Technology. AVI Publishing Company, INC. Westport. Oyen, L.P.A. & Nguyen, X. D. (1999). Plant Resources of South-East Asia No. 19. Essential oil Plants. Backhuys Publishers. Leiden. Parker, S.P. (1993). Encyclopedia of Chemistry 2nd Edition. McGraw-Hill, New York. Sun, R. Zhang, Q. Wang, B. Xu, (1993). Programmed temperature gas chromatography retention index. J. Chromatogr A. 657, 1-15 www.pinechemicals.biz, 2002 Zubrick, J.W. (1984). The Organic Chem. Lab. Survival Manual: A Student’s Guide To Techniques. John Wiley & Son. New York.

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Table 4. Retention indices of hydro distilled essential oils for cocoa branches and leaves for both cropping season and location

Chemical compound UIT1 NA33 UIT1 NA33 UIT1 NA33 UIT1 NA33 UIT1 NA33 UIT1 NA33 UIT1 NA33 UIT1 NA33 Average Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Branches Branches Branches Branches Branches Branches Branches Branches H/Perak H/Perak Tawau Tawau Tawau Tawau H/Perak H/Perak Tawau Tawau H/Perak H/Perak Tawau Tawau H/Perak H/Perak Off Off Off Off Peak Peak Peak Peak Off Off Off Off Peak Peak Peak Peak 2-hexenal* 1233 1235 1236 - 1237 - 1235 1234 ------1235 6-methyl-5-hepten-2-one 1347 - 1346 - - 1345 1345 1346 ------1346 Hexanol - - - - - 1350 1351 1351 ------1351 (Z)-3-hexenol 1380 1383 1383 1382 1382 1385 1382 1382 1383 1383 - 1385 1384 1384 1383 1383 1383 linalool oxide* 1437 1436 1438 1437 1437 1436 1435 1435 1438 1437 1438 1436 1436 1438 1437 1438 1437 linalool oxide* 1463 1463 1464 1462 1463 - 1463 - 1462 1462 1464 1464 1465 1463 1462 1462 1463 Benzaldehyde 1516 1517 1517 - - - - - 1516 1516 1515 1514 1516 - - 1518 1516 linalool 1536 1537 1538 1536 1536 1538 1538 1537 1536 1536 1538 1538 1537 1537 1538 1536 1537 6-methyl-3,5-heptadien-2-one 1583 1584 - - - - 1583 1583 ------1583 Terpinen-4-ol ------1608 - - - 1607 - 1608 benzenaacetaldehyde ------1642 1642 1642 - 1641 - 1642 2-hydroxy-benzaldehyde ------1664 - - - 1664 terpineol - 1679 1680 1678 1678 1680 1679 1678 1679 1678 1680 1680 1680 1678 1678 1678 1679 epoxylinalol* 1721 1721 - - 1720 - 1720 - 1721 1721 1718 1718 1721 1719 1720 1720 1720 epoxylinalol* - - 1745 1745 1745 - - - - - 1746 1744 - 1742 - 1745 1745 methyl salicylate 1756 1757 - 1755 - - 1756 1756 1758 1758 1756 1754 - - - 1755 1756 Nerol - - - 1780 ------1781 - 1782 - - - 1781 damascenone 1795 1796 1796 1797 1797 1795 1798 - 1796 ------1796 ionone* 1826 1826 1827 1825 1823 1826 1828 1828 - - - 1824 - - - - 1826 geranyl acetone - 1829 - - 1828 ------1827 - - - - 1828 Geraniol - - - 1831 - - - - - 1830 - - 1830 1832 1828 - 1830 hexanoic acid ------1833 - - 1833 Nerilacetone - - - - - 1837 ------1837 2-methoxy-phenol ------1840 - - - - - 1841 - - - 1841 Benzylalcohol 1855 1855 1859 1858 1855 1857 1857 1857 1856 1857 1856 1857 1858 - 1856 1856 1857 2,6-bis-(1,1-dimethylethyl)-4- - - - - 1886 ------1886 methylphenol Benzeethanol 1889 1889 1886 1886 - 1888 1887 - 1889 1889 1887 1887 1889 1888 - 1888 1888 ionone 1908 1909 1907 1908 1909 1908 1907 1908 ------1908 (-)-(5R,6S)-5,6-epoxy-5,6- - - - - 1955 1957 ------1956 dihydro-b-ionone Tetradecanal - - - - 1988 ------1988 (E)-nerolidol - - - - - 2005 2008 ------2007 octanoic acid ------2036 2036 - - - 2036 - - - 2036 -nonanoic lactone 2037 ------2036 - - - - - 2037 6,10,14-trimethyl-2- 2084 - - - 2083 2085 2084 2083 ------2084 pentadecanone nonanoic acid ------2088 ------2088 (E,E)-pseudoionone 2099 2099 ------2099 Eugenol 2131 2129 2130 - 2131 ------2132 - - - 2131 4-ethoxy-ethylester-benzoic ------2158 2158 acid

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4-ethoxy-benzoic acid ------2161 - - - - - 2161 decanoic acid 2188 2187 - - - - - 2188 2187 2188 2189 - - - 2188 - 2188 2,4-bis-(1,1- - 2283 - - 2282 - - 2282 - - - 2281 2282 - - - 2282 dimethylethyl)phenol 2-methoxy-4-(1------2300 - 2299 2299 - - 2301 - - - 2300 propenyl)phenol 4-vinyl-2-methoxy-phenol - - 2313 2313 ------2313 farnesyl acetone* 2322 2322 2323 2323 2320 2323 2322 2323 ------2322 Isoeugenol ------2363 2363 - - - - 2363 Indole 2436 - 2436 ------2436 3,4-dihydro-1H-2------2456 2457 - - - - 2457 benzopyran-1-one dodecanoic acid 2468 2468 2469 2467 - - 2467 2466 2468 2468 2470 2468 2468 2469 2466 2469 2468 Farnesol* 2479 2475 - 2477 2476 2478 - 2477 ------2477 Phytol 2550 2552 2551 2551 2550 2551 2550 2551 ------2551 di-n-butylphathalate 2634 - 2638 ------2636 tetradecanoic acid 2644 2644 2644 2642 - 2642 - 2643 2642 2643 2643 2647 2642 2643 2644 2641 2643 pentadecanoic acid 2742 2742 - - - - - 2740 2739 2739 2744 2740 - 2740 2738 2740 2740 2-hydroxy------2771 2771 2770 2769 - 2769 2770 cyclopentadecanone 14-pentadecenoic acid ------2772 2772 ------2772 hexadecanoic acid 2888 2889 2888 2891 2889 2890 2890 2891 2890 2887 2891 2890 - 2891 - 2890 2890 (+/-)-15-hexadecanolide ------2929 ------2929 9-hexadecenoic acid ------2932 - 2930 - - 2931 - 2931 2931 linoleic acid 2947 2948 ------2947 2948 2949 2946 - 2949 - - 2948 * correct isomeric form not identified

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Table 5. Percentage peak area of hydro distilled essential oils for cocoa branches and leaves for both cropping season and location

Chemical compound UIT1 NA33 UIT1 NA33 UIT1 NA33 UIT1 NA33 UIT1 NA33 UIT1 NA33 UIT1 NA33 UIT1 NA33 Leaves Leaves Leaves Leaves Leaves Leaves Leaves Leaves Branches Branches Branches Branches Branches Branches Branches Branches H/Perak H/Perak Tawau Tawau Tawau Tawau H/Perak H/Perak Tawau Tawau H/Perak H/Perak Tawau Tawau H/Perak H/Perak Off Off Off Off Peak Peak Peak Peak Off Off Off Off Peak Peak Peak Peak 2-hexenal* 1.12 0.80 0.82 - 3.55 - 1.04 2.42 ------6-methyl-5-hepten-2-one 0.54 - 0.82 - - 1.14 0.88 2.17 ------Hexanol - - - - - 3.01 2.11 0.5 ------(Z)-3-hexenol 5.07 2.33 2.31 3.92 7.44 19.79 9.3 2.66 0.42 0.58 - 1.03 0.72 1.18 1.73 2.47 linalool oxide* 1.7 2.83 1.92 2.85 11.48 1.76 1.72 1.42 7.43 10.19 13.27 15.32 12.93 10.29 24.5 11.17 linalool oxide* 2.2 2.54 2.24 2.97 10.55 - 4.96 - 6.76 7.00 13.29 10.85 11.31 6.79 19.37 7.68 Benzaldehyde 2.26 1.98 0.86 - - - - - 0.77 0.73 1.68 1.94 2.48 - - 1.55 linalool 2.42 3.06 5.68 7.93 4.81 5.71 6.15 1.86 4.99 4.30 6.89 3.8 11.3 8.25 12.41 4.8 6-methyl-3,5-heptadien-2-one 0.65 2.42 - - - - 1.86 6.97 ------Terpinen-4-ol ------2.11 - - - 4.43 - benzenaacetaldehyde ------1.27 1.31 1.7 - 1.96 - 2-hydroxy-benzaldehyde ------0.98 - - - terpineol - 1.72 2.36 5.09 1.49 1.79 2.22 0.59 2.48 4.90 5.58 5.72 7.5 4.99 5.41 3.5 epoxylinalol* 1.13 1.86 - - 4.52 - 2.95 - 6.29 6.80 9.54 10.37 7.09 6.74 10.64 8.07 epoxylinalol* - - 2.29 3.38 5.93 - - - - - 10.99 9.58 - 6.42 - 9.01 methyl salicylate 0.7 0.56 - 1.93 - - 1.81 0.74 0.94 1.04 1.64 4.08 - - - 5.52 Nerol - - - 1.07 ------0.74 - 1.26 - - - damascenone 1.58 1.16 1.29 2.23 0.65 1.77 1.87 - 0.46 ------ionone* 3.6 2.90 3.03 2.59 3.84 5.54 2.08 1.78 - - - 1.23 - - - - geranyl acetone - 4.37 - - 2.71 ------1.54 - - - - Geraniol - - - 3.29 - - - - - 1.45 - - 3.83 1.67 1.47 - hexanoic acid ------1.70 - - Nerilacetone - - - - - 10.26 ------2-methoxy-phenol - 0.54 - - - - 0.55 - - - - - 0.75 - - - Benzylalcohol 1.89 1.71 2.85 2.97 2.08 1.87 2.34 1.3 1.01 0.86 2.18 2.53 2.47 - 2.00 3.69 2,6-bis-(1,1-dimethylethyl)-4- - - - - 1.24 ------methylphenol Benzeethanol 1.49 2.02 1.58 1.09 - 2.36 2.42 - 1.83 2.68 0.76 0.94 3.26 1.76 - 2.16 ionone 1.96 1.09 1.96 1.63 0.96 2.39 1.17 1.35 ------(-)-(5R,6S)-5,6-epoxy-5,6- - - - - 1.21 1.72 ------dihydro-b-ionone Tetradecanal - - - - 1.89 ------(E)-nerolidol - - - - - 4.17 0.86 ------Octanoic acid ------1.41 0.96 - - - 1.00 - - - -nonanoic lactone 1.3 ------0.75 - - - - - 6,10,14-trimethyl-2- 3.17 - - - 3.94 2.85 3.65 4.91 ------pentadecanone nonanoic acid ------0.53 ------(E,E)-pseudoionone 0.89 0.80 ------Eugenol 2.56 2.35 1.23 - 1.30 ------2.7 - - - 4-ethoxy-ethylester-benzoic ------1.82 acid

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4-ethoxy-benzoic acid ------3.2 ------decanoic acid 0.55 0.54 - - - - - 1.11 0.83 0.41 0.69 - - - 2.42 - 2,4-bis-(1,1- - 1.51 - - 5.01 - - 1.09 - - - 0.89 1.24 - - - dimethylethyl)phenol 2-methoxy-4-(1------3.16 - 1.65 0.78 - - 2.37 - - - propenyl)phenol 4-vinyl-2-methoxy-phenol - - 1.55 1.2 ------Farnesyl acetone* 3.57 4.01 2.21 5.8 1.88 3.66 4.59 4.13 ------Isoeugenol ------1.35 0.94 - - - - Indole 0.66 - 0.9 ------3,4-dihydro-1H-2------1.34 1.69 - - - - benzopyran-1-one dodecanoic acid 1.94 2.22 2.35 2.11 - - 0.85 1.32 1.51 1.29 3.13 3.65 2.34 1.39 2.42 4.51 Farnesol* 2.69 1.32 - 1.39 1.44 1.85 - 0.84 ------Phytol 2.19 2.79 2.99 3.4 1.88 2.08 2.92 1.03 ------di-n-butylphathalate 0.6 - 0.82 ------tetradecanoic acid 1.56 1.56 2.44 2.11 - 1.08 0.72 1.7 3.49 2.61 2.21 2.96 4.06 1.66 1.8 3.74 pentadecanoic acid 0.63 0.52 - - - - - 0.46 2.93 2.25 1.2 1.45 - 1.51 1.31 1.72 2-hydroxy------1.02 1.18 1.1 1.61 - 1.81 cyclopentadecanone 14-pentadecenoic acid ------3.61 2.5 ------hexadecanoic acid 9.00 9.77 25.74 21.09 4.83 4.21 2.92 5.94 18.34 19.25 9.44 12.68 - 23.55 - 19.23 (+/-)-15-hexadecanolide ------3.35 ------9-hexadecenoic acid ------3.04 - 0.67 - - 2.39 - 1.56 linoleic acid 0.85 0.72 ------9.99 1.35 1.87 2.65 - 1.76 - - * correct isomeric form not identified

Malaysian Cocoa Journal 88

Short communication

TAXONOMIC STATUS OF TRICHOGRAMMATOIDEA BACTRAE FUMATA NAGARAJA, AN EGG PARASITOID OF THE COCOA POD BORER, CONOPOMORPHA CRAMERELLA (SNELLEN) IN MALAYSIA: A CRITICAL REVIEW

Alias A.1, I. Azhar1 and M. Schilthuizen2 Malaysian Cocoa Board, Locked Bag 211, 88999 Kota Kinabalu, Sabah1 Universiti Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu, Sabah2

Malaysian Cocoa J. 1: 83-85 (2004) Numerous studies have been made to search the potential natural enemies of the cocoa pod borer (CPB), Conopomorpha cramerella (Snellen) as alternative control method since the pest was reported attacking cocoa in 1980. This includes egg and pupal parasitoids. In Sabah, Lim (1983) discovered an indigenous egg parasitoid parasitizing on CPB eggs, which was identified as Trichogrammatoidea bactrae fumata Nagaraja. Since then, various studies have been conducted to evaluate the effectiveness of the parasitoids both in the laboratory and in the fields (Lim and Chong, 1987; Lee et al., 1995). In general, the efficacy of Td. bactrae fumata for controlling the CPB was initially slow, but gradually improved thereafter to provide a good control.

Three year later, the specimens of Td. bactrae fumata were re-examined and found to be erroneously identified. The species was found to be Trichogrammatoidea sp. near cojuangcoi Nagaraja, as it was observed to be closely related to Trichogrammatoidea cojuangcoi Nagaraja from the Philippines and Indonesia (Nagaraja et al., 1985; Nagaraja, 1985)). Evidence from hybridization studies using species from Sabah, Philippines and Indonesia showed that the egg parasitoids do not interbreed freely (Nagaraja et al., 1985). This can be seen from the small number of progeny produced and it suggests that the populations are of extremely limited compatibility. In view of that, most taxonomists would normally considered such populations sibling species (Nagaraja, 1985). This finding supports the evidence that the earlier identification of Td. bactrae fumata is inaccurate. Reports on sibling species of Trichogramma (Nagarkatti and Nagaraja, 1977) and Trichogrammatoidea (Nagaraja, 1978) have been documented elsewhere. In case of Td. sp. near cojuangcoi, this species may eventually become totally reproductive isolated whereby the populations will diverge to evolve as full species in the future.

A survey in 2004 revealed Td. cojuangcoi parasitizing on CPB eggs in Tawau, Semporna and Tenom, Sabah, Malaysia. The absence of Td. sp. near cojuangcoi from cocoa is questionable and suggests that it might be wrongly identified since it was closely related to and indistinguishable from Td. cojuangcoi. Since no hybridization study was undertaken, it can be assumed that Td. sp. near cojuangcoi is also referrable to Td. cojuangcoi, and both belong to the same species (Nagaraja, pers. comm., 2004).

Today, many researchers still use the earlier taxon name although rectification on the identity was made in 1985 (Nagaraja et al., 1985, Nagaraja, 1985). This may be due to their being unaware about the changes of the taxon name or to some disagreement about the status of the parasitoid (Ooi, 1987). But as shown by Nagaraja et al (1985) and recent studies, the latest taxon name should be used since it was suggested by the same taxonomist who identified Td. bactrae fumata.

Malaysian Cocoa Journal 89

Plate 1. Female of Trichogrammatoidea Plate 2. Male of Trichogrammatoidea cojuangcoi cojuangcoi

Plate 3. Antenna of female Td. Cojuangcoi Plate 4. Antenna of male Td. Cojuangcoi

Absent in Rs1 Absent in Rs1

Plate 5. Fore wing of female Td. cojuangcoi [Rs1 Plate 6. Fore wing of male Td. cojuangcoi [Rs1 – – Radial sector (vein tract-1st abscissa)] Radial sector (vein tract-1st abscissa)]

Malaysian Cocoa Journal 90

ACKNOWLEDGEMENTS

The authors would like to Mr. Kelvin Lamin, Director of Biology Division for his constructive comments on this manuscript. Appreciation is due to Dr. H. Nagaraja of Bangalore, India for his kindness to identify the Trichogrammatoidea species.

REFERENCES

Lee, C.T., E.B. Tay, & M.T. Lee. 1995b. The use of TBF for the management of cocoa pod borer. Paper presented at the Workshop on Recent Advances in the Management of Cocoa Pod Borer (With special reference to TBF), Marco Polo Hotel, Tawau, Malaysia. June 29, 1995. Lim, G. T. 1983. Trichogrammatoidea bactrae fumata Nagaraja, a new egg parasitoid of cocoa pod borer. Kuala Lumpur. Proc. Seminar on Advance in Plant Protection in Malaysia. pp12-13. Lim, G.T. & T.C. Chong 1987. Biological control of cocoa pod borer, Conopomorpha cramerella Snellen by periodic release of Trichogrammatoidea bactrae fumata Nagaraja in Sabah, Malaysia. In: Management of cocoa pod borer. (Edited by Ooi, P.A.C., Chan, L.G., Khoo, K.C., Teoh, C.H., Jusoh, M.M., Ho, C.T. and Lim, G.S.). Malaysian Plant Protection Society, Kuala Lumpur. pp71-80. Nagarkatti, S. and Nagaraja, H. 1977. Biosystematics of Trichogramma and Trichogrammatoidea species. Annual Review of Entomology 22, 157-176. Nagaraja, H. 1978. Experimental hybdridization between some species of Trichogrammatoidea Girault (Hymenoptera, Trichogrammatidae). Journal of Entomological Research 2, 192-198. Nagaraja, H., Wordojo, S., Reyes, T.M., Easaw, P.T. and Vanialingam, T. 1985. Record of egg parasite of cacao pod-borer in Indonesia. Planter 61, 469-472. Nagaraja, H. 1985. Description of a new species of Trichogrammatoidea (Hymenoptera, Trichogrammatidae), parasitic on the cacao pod borer in the Philippines. Philippine Entomologist 6, 207-213. Ooi, P.A.C. 1987. Advances in the biological control of cocoa pod borer. (Edited by Ooi, P.A.C., Chan, L.G., Khoo, K.C., Teoh, C.H., Jusoh, M.M., Ho, C.T. and Lim, G.S.). Malaysian Plant Protection Society, Kuala Lumpur. pp103-117.

Malaysian Cocoa Journal 91

Short communication

NEW RECORD OF TRICHOGRAMMA CHILONIS ISHII (HYMENOPTERA: TRICHOGRAMMATIDAE) AS AN EGG PARASITOID OF THE COCOA POD BORER, CONOPOMORPHA CRAMERELLA (SNELLEN) (LEPIDOPTERA: GRACILLARIIDAE) IN MALAYSIA

Alias A. 1, M. Schilthuizen2 and H. Sulaiman1 Malaysian Cocoa Board, Locked Bag 211, 88999 Kota Kinabalu, Sabah1 Universiti Malaysia Sabah, Locked Bag 2073, 88999 Kota Kinabalu, Sabah2

Malaysian Cocoa J. 1: 86-88 (2004) Trichogrammatoidea sp. near cojuangcoi Nagaraja is an indigenous egg parasitoid in Sabah, Malaysia, first reported to parasitize eggs of the cocoa pod borer (CPB), Conopomorpha cramerella (Snellen) in 1982 (Lim 1983; 1986). However, it was initially erroneously identified as Trichogrammatoidea bactrae fumata Nagaraja (Nagaraja et al., 1985; Nagaraja, 1985) and most of the early literature on this system uses the latter taxon name. The efficacy of the parasitoids for controlling the CPB was demonstrated and confirmed (Lim and Chong, 1987; Lee et al., 1995). Until today, it is the only species that has been used in the mass production and release for CPB control. According to Hassan (1994), the efficacy of egg parasitoid for controlling insect pests can be improved if more than one species are released simultaneously. However, the choice of combination should be based on the results of laboratory, semi-field and field experiments. Therefore, a survey was conducted to search additional egg parasitoids of CPB throughout Sabah.

During the survey, an egg parasitoid, Trichogramma chilonis Ishii was found parasitizing on CPB eggs in the cocoa fields in Tawau, Semporna, Kunak, Kota Marudu, Keningau and Tenom, Sabah, Malaysia (Plate 1 – 6). This is the first record in Malaysia of T. chilonis parasitizing on CPB eggs. T. chilonis has also been referred to as Trichogramma confusum Viaggiani, a junior synonym (Nagarkatti and Nagaraja, 1979). The parasitoid was regards as ubiquitous and distributed throughout Southeast Asia, China, Taiwan, Japan and Papua New Guinea. In Malaysia, T. confusum was reported attacking the eggs of sugarcane borer, Tetramoera schistaceana (Lim and Pan, 1980).

Under laboratory condition, T. chilonis completes its life cycle from egg to adult emergence in 7.8 ± 0.6 days on C. cramerella eggs and 7.1 ± 0.3 days on the rice moth eggs (Corcyra cephalonica Stainton). The longevity of T. chilonis starved and 10% honey-fed was 1.5 ± 0.7 days and 1.9 ± 0.6 days, respectively. The sex ratio of T. chilonis on its original host and Corcyra eggs were 1:1.3 (♂:♀) and 1:1.8 (♂:♀), respectively. In the laboratory, each parasitised Corcyra eggs produced 1-2 parasitoids, while only one parasitoid was ever observed emerging from CPB egg because it is smaller in size. Unfertilized females produce male offspring while fertilized females produce both sexes. The field rate of parasitism on cocoa pod borer eggs varied from 26.1 - 69.8% with an average of 52.9 ± 16.8%.

T. chilonis can be easily mass-produced using Corcyra eggs, thus making it a potential biocontrol agent against the CPB. However, further study need to be carried out to evaluate the efficacy of T. chilonis for controlling the CPB before it is mass-produced and released. Such a study should includes (i) the density of T. chilonis to be released, (ii) release, timing and frequency of T. chilonis, and (iii) other factors that influence the efficacy of T. chilonis in cocoa fields including its compatibility with the existing parasitoids.

Malaysian Cocoa Journal 92

Plate 1. Female of Trichogramma chilonis Plate 2. Male of Trichogramma chilonis

Plate 3. Antenna of female T. chilonis Plate 4. Antenna of male T. chilonis

Plate 5. Fore wing of female T. chilonis Plate 6. Fore wing of male T. chilonis

Malaysian Cocoa Journal 93

ACKNOWLEDGEMENTS

The authors would like to thank the Director General of the Malaysian Cocoa Board for his permission to publish this paper and Mr. Kelvin Lamin, Director of Biology Division for his constructive comments. Appreciation is due to Dr. H. Nagaraja of Bangalore, India for his kindness to identify the Trichogramma species.

REFERENCES

Hassan, S.A. 1994. Strategies to select Trichogramma species for use in biological control. In: Biology of egg parasitoids (Edited by E. Wajnberg and S.A. Hassan). CAB International. Wallingford. UK. pp55-71. Lee, C.T., E.B. Tay, & M.T. Lee. 1995. The use of TBF for the management of cocoa pod borer. Paper presented at the Workshop on Recent Advances in the Management of Cocoa Pod Borer (With special reference to TBF), Marco Polo Hotel, Tawau, Malaysia. June 29, 1995. Lim, G.T. and Pan, Y.C. 1980. Entomofauna of sugarcane in Malaysia. Proc. XVII ISST Congr., Manila, Phillipines. pp1658-1679. Lim, G. T. 1983. Trichogrammatoidea bactrae fumata Nagaraja, a new egg parasitoid of cocoa pod borer. Kuala Lumpur. Proc. Seminar on Advance in Plant Protection in Malaysia. pp12-13. Lim, G.T. 1986. Biological studies on Trichogrammatoidea bactrae fumata Nagaraja in the laboratory. Journal Application of Entomology 101,48-54. Lim, G.T. & T.C. Chong 1987. Biological control of cocoa pod borer, Conopomorpha cramerella (Snellen) by periodic release of Trichogrammatoidea bactrae fumata Nagaraja in Sabah, Malaysia. In: Management of cocoa pod borer. (Edited by Ooi, P.A.C., Chan, L.G., Khoo, K.C., Teoh, C.H., Jusoh, M.M., Ho, C.T. and Lim, G.S.). Malaysian Plant Protection Society, Kuala Lumpur. pp71-80. Nagarkatti, S. and Nagaraja, H. 1979. The status of Trichogramma chilonis Ishii (Hym.: Trichogrammatidae). Oriental insects 13(1-2), 115-118. Nagaraja, H., Wordojo, S., Reyes, T.M., Easaw, P.T. and Vanialingam, T. 1985. Record of egg parasite of cacao pod-borer in Indonesia. Planter 61, 469-472. Nagaraja, H. 1985. Description of a new species of Trichogrammatoidea (Hymenoptera, Trichogrammatidae), parasitic on the cacao pod borer in the Philippines. Philippine Entomologist 6, 207-213.