Sugarcane: Research Towards Efficient and Sustainable Production. Wilson JR, Hogarth D M , Campbell JA and Garside AL (Eds).
CSIRO Division of Tropical Crops and Pastures, Brisbane. 1996. p p . 111-113 111 POTENTIAL FOR INCREASING SUGAR PRODUCTIVITY THROUGH BIOTECHNOLOGY IN MAURITIUS
DOOKUN A, D O M A I N G U E R and SAUMTALLY S
Mauritius Sugar Industry Research Institute Reduit Mauritius
A B S T R A C T
During the last decade, sugar production in Mauritius has faced constraints such as unfavourable climatic conditions, increasing cost of labour, loss of land under sugarcane to urbanization, and competition from new industries. To meet the requirement of some 0.65Mt sugar, the country has to adopt new technologies to increase production efficiency per unit area. Biotechnology is expected to play a major role in increasing this efficiency. New techniques of disease diagnosis, such as monoclonal antibodies, DNA probes and the polymerase chain reaction should enable increased sensitivity, speed and reliability of pathogen detection. Monoclonal antibodies and DNA probes have been produced for the diagnosis of the gumming disease pathogen (Xanthomonas campestris pv.
vasculorum). These techniques are also being used to study leaf scald bacterium (Xanthomonas albilineans), to differentiate the African and Mascarene serotypes that exist in Mauritius, and their use will be extended to other pathogens. These new diagnostic tools will be important in the safe international movement of germptasm, in the characterization of pathogen variants and in epidemiological studies to allow more efficient control measures to be formulated. Another advance is expected through the in vitro culture of sugarcane and the micropropagalion of new varieties using this method on a large scale. Coupled with new diagnostic techniques, tissue culture will speed the release of varieties free from diseases to the planting community. Genetic fingerprinting of varieties, using restriction fragment length polymorphism (RFLP) analysis and random amplified polymorphic DNA (RAPD), is being carried out. This should allow a better choice of parents for crossing and hence make the breeding programme more efficient.
Molecular markers associated with two important fungal diseases, rust (Puccinia melanocephala) and yellow spot (Mycovellosiella koepkei) by the RAPD technique are being sought. This approach will aid the rapid screening of varieties at an early stage in the selection programme, with the aim of producing varieties specifically adapted to the wet uplands of Mauritius, where resistance to both rust and yellow spot is essential.
INTRODUCTION
Sugarcane is the most important crop in Mauritius, contributing about 3 0 % of the i s l a n d ' s gross export e a r n i n g s and providing m o r e employment than any other industry. Sugarcane is well adapted to the local soil and climatic conditions and tolerates cyclonic winds. Such characteristics have brought about its extensive cultivation for more than a century, and Mauritius is one of the world's most efficient producers. Presently, the sugar industry is facing several difficulties : a rapid loss of cane lands to urbanization; scarcity of labour; increases in production costs; low prices on the world market; and competition with new industries. Despite these difficulties, the industry has the will to increase its sugar productivity. Several measures have been proposed to meet this objective. Research will play an important role in providing better varieties, and biotechnology has been identified as one means that could contribute to increased sugar production. Various aspects of biotechnology can be applied to sugarcane. These include tissue culture, genetic t r a n s f o r m a t i o n , i m p r o v e d d i s e a s e d i a g n o s t i c tools and application of molecular markers to plant breeding. This paper reports some of the biotechnology approaches being followed by the Mauritius Sugar Industry Research Institute (MSIRI).
TISSUE CULTURE
At least one new improved variety is released annually in Mauritius. At release, the material available in nurseries is often inadequate to supply all planters willing to exploit the variety immediately. Micropropagation by in vitro culture of new varieties would help to obtain plantlets rapidly for large scale cultivation and also ensure clean, disease-free material (De Boer & Rao 1991). Hot water treatment of setts prior to the establishment of nurseries will also not be required. After identifying promising varieties, cultures are established in vitro from apical buds and meristems and at the final stages of selection, they can be rapidly bulked. At MSIRI with two laminar flow cabinets, one growth room, glasshouse space, two technicians and one scientist, we can produce 150.000 plantlets of a new variety in 8 months, according to the scheme described in Fig. 1. This material will be sufficient to plant 10 ha of nurseries. These facilities may be expanded to produce more material.
Further research is needed on the conditioning and transplanting of plantlets into commercial fields.
Through tissue culture techniques, sugarcane calli can be produced for genetic transformation by bombardment with a biolistic gun (Bower &
Apical bud or meristem culture •
i
4 weeks 50 plantlets Transfer to multiplication medium 4 weeks »- 50 clusters Splitting and subcultured on multiplication medium 3
M I
T j. 4 weeks .. 250 plantlets m
g 4 weeks • 1 250 plantlets n
p J h
R 4 weeks ,. 6 250 plantlets s
P i I A 4 Weeks „ 31 250 plantlets
I A V
4 | 4 weeks „ 156 250 plantlets
O i N t Glasshouse Rooting ^ 14 weeks Fig. 1 Multiplication scheme for the in vitro micropropagation of
sugarcane.
Birch 1992). This approach is being investigated at MSIRI for the introduction of herbicide resistance genes into sugarcane varieties. At a later stage, the introduction of genes conferring resistance to diseases would be contemplated.
DIAGNOSTIC TOOLS
Diseases may be a serious constraint to productivity and an early and accurate diagnosis is important. Bacterial diseases such as gumming, leaf scald, and, to a lesser extent, ratoon stunting have caused economic losses in Mauritius. It is thus desirable to reinforce conventional
112
detection methods for these diseases with biotechnology techniques.
New identification methods would increase speed and sensitivity of diagnosis, and generate information that would provide a better understanding of the epidemiology of the pathogen and hence the most suitable control measures to be adopted.
G u m m i n g disease (Xanthomonas campestris pv vasculorum) is a dangerous threat to Mauritius and the Mascarene region. Three races of the bacterium occur in Mauritius and different entities of the pathogen are suspected to exist in the Mascarene and Southern African region (Qhobela & Claflin 1992). In 1964 and 1980, compulsory uprooting of two major varieties was enforced by law as they had succumbed to new races of the bacterium. The development of monoclonal antibodies in Mauritius provided the necessary tool to investigate variation in the pathogen. Isolates from South Africa, Zimbabwe, Madagascar and Mauritius were shown to be distinct using monoclonals (Dookun 1993).
Moreover, in Mauritius, race 1 proved to be serologically different from races 2 and 3. A genetic study of the same isolates by RFLP analysis confirmed this heterogeneity of the gumming pathogen on a local and r e g i o n a l b a s i s ( S S a u m t a l l y , u n p u b l i s h e d d a t a ) . T h e s e s t u d i e s demonstrate the high variability in the population of the bacterium, the possible emergence of new races and the danger of introducing new strains from neighbouring regions. Specific genetic differences among the isolates, such as the presence of a ubiquitous plasmid in race 1, is being exploited for detection by the polymerase chain reaction (PCR).
Outbreaks of leaf scald (Xanthomonas albilineans) have occurred recently in several countries, including Mauritius. These epidemics have led to the rejection of several commercial clones and variation in the pathogen has been suggested as an explanation for the outbreaks. In Mauritius, two serotypes (Mascarene & African) of the bacterium have been detected (Autrey et al 1995). To investigate the epidemiology of the disease, RFLP analysis of the isolates has been conducted using DNA probes produced by genomic subtraction. The two serotypes were found to be genetically different (Y Fakim & A Dookun, unpublished data) and DNA sequencing of specific DNA fragments is being carried out for the eventual development of primers for detection by PCR. This will allow the distribution of the two variants to be determined as well as producing a rapid test to ensure that planting material is disease- free.
The exchange of sugarcane plant material between countries needs to be tightly regulated owing to several systemic diseases. Disease diagnosis techniques based on molecular detection technology will be valuable tools for the early detection of diseases during quarantine. A reliable technique is essential because glasshouse conditions (high humidity, temperature fluctuations, lack of sunshine) often mask disease symptoms. Biotechnology tools such as PCR offer a high level of detection in the absence of symptoms. The necessity for extreme caution in germplasm exchange has been amplified in recent years with the discovery of several new diseases.
M O L E C U L A R M A R K E R S
Plant breeders and plant pathologists require accurate screening methods to help them release varieties with specific phenotypic characters.
Varietal screening against major diseases has often proved difficult as factors such as climatic conditions, physiological status of the plant and fluctuations in disease pressure are involved. Variety x year interactions have been observed, necessitating a lengthy screening p r o g r a m . M o l e c u l a r m a r k e r s would help to identify the g e n e s responsible for a specific phenotype to a region of the genome. Provided that the markers were not cross-specific, and depending on how closely they were associated, the association of such markers in the progeny would enhance the efficiency of the selection process.
For the two important fungal diseases, rust (Puccinia melanocephala) and yellow spot (Mycovellosiella koepkei), the search for molecular markers has been initiated using the random amplified polymorphic DNA (RAPD) technique. Progenies derived from several biparental crosses and selfing of parents with known rust resistance, have been evaluated in the field and in the laboratory. D N A extracts from the different populations are being studied in the search for markers that
could be associated with the disease resistance gene(s). Recent findings revealed that three to four genes are involved in the resistance to yellow spot disease in a dominant way (Ramdoyal et al 1996, Table 1).
Fingerprinting of varieties would also allow genetic diversity to be examined and might provide a method for clonal classification.
Table 1 Probable genetic constitution of different phenotypes in relation to yellow spot disease : (a) 3 gene pair; and (b) 4 gene pair. [R
= resistant, SS = slightly susceptible, S = susceptible, HS = highly susceptible)
(a)
Phenotype Genotype Gene 1 Gene 2 Gene 3
(At least 3 dominant genes, one at each locus) (At least 2 dominant genes, one at any two loci) (At least 1 dominant gene, at only one locus) (Absence of dominant genes)
(At least 3 dominant genes, one at any two loci) (At least 2 dominant genes, one at any two loci) (At least 1 dominant gene, at only one locus) (Absence of dominant genes) PERSPECTIVES
The application of biotechnology to sugarcane is still in its embryonic stage, but our knowledge and technical capabilities are rapidly increasing. Thus, the genetic transformation which was believed to be successful only in the long term is now a reality. It is therefore important to explore the various avenues offered by biotechnology in order to reap the full benefits. With this objective, the MSIRI launched its biotechnology program in 1993. Tissue culture and disease diagnosis t e c h n i q u e s are already m e e t i n g the desired objectives. In vitro micropropagation is now enabling the early exploitation of newly released varieties to growers. Tools for disease detection are allowing unequivocal identification and detection of low levels of infection of several diseases, and are already generating information on pathogen variation and a better understanding of the epidemiology of diseases.
Mid-term perspectives include the study of the genome of Mauritian sugarcane clones to help the improvement of varieties in a more efficient manner. Linking of molecular markers to agronomic traits relevant to the country is also a major goal of the program. However, in order to make significant progress over a short period, it is imperative to establish collaborative relationships with other institutions to strengthen our research capacities.
A C K N O W L E D G E M E N T S
We thank Dr R Julien, Director, Mauritius Sugar Industry Research Institute for reviewing the manuscript and for his permission for publication.
R = R1 R2 R3
S S = r1 R2 R3
S = R1 r2 r3
H S = r1 r2 r3
(b)
Phenotype Genotype Genel Gene 2 Gene 3 Gene 4
R = R, R, R3 R3
S S = r, r2 R3 R3
S = R, r2 r3 r3
H S = r, r2 r3 r3
R E F E R E N C E S
Autrey LJC, Saumtally S, Dookun A, Medan H (1995) Studies on variation in the leaf scald pathogen Xanthomonas albilineans.
Proceeding of the International Society of Sugar Cane Technologists. XXI, 485 - 497.
B o w e r R , Birch R G ( 1 9 9 2 ) T r a n s g e n i c s u g a r c a n e p l a n t s via microprojectile bombardment. Plant Journal 2, 409-416.
De Boer H, Rao S (1991) Disease free sugarcane nursery through tissue culture. 9th Annual Barbados Sugar Technologists'Association, 57- 60.
D o o k u n A (1993) T h e p r o d u c t i o n , c h a r a c t e r i z a t i o n and use of
113 monoclonal antibodies for race differentiation of Xanthomonas campestris pv vasculorum causal agent of gumming disease of sugarcane. PhD thesis. University of Reading, 212 pp.
Qhobela M, Claflin LE (1992) Eastern and Southern African strains of Xanthomonas campestris pv vasculorum are distinguishable by r e s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m o f D N A a n d polyacrylamide gel electrophoresis of membrane proteins. Plant Pathology 4 1 , 113-121.
Ramdoyal K, Domaingue R, Sullivan S and Autrey LJC (1996) Studies on the inheritance of yellow spot (Mycovellosiella koepkei) disease in sugarcane. Proceeding of the International Society of Sugar Cane Technologists. XXII (in press).
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Sugarcane: Research Towards Efficient and Sustainable Production. Wilson JR, Hogarth D M , Campbell JA and Garside AL (Eds).
114 C S I R O Division of Tropical Crops and Pastures, Brisbane. 1996. pp. 114-116
BIOTECHNOLOGY IN THE SUGAR INDUSTRY: SOCIO-ECONOMIC ASPECTS OF PROBLEMS AND PROSPECTS FOR DEVELOPING COUNTRIES
SINGH S
CARON1 (1975) LIMITED, Brechin Castle, Couva, Trinidad, W.I.
ABSTRACT
Within the last decade, the emergence of biotechnology and related developments have spawned fertile grounds which now offer and will no doubt continue to offer inexpensive and speedy solutions to fundamental agricultural and agro-industrial problems.
These solutions can be applied in all latitudes with equal effectiveness and possibly with equal benefits. Results to date are indicative of the potential available to broaden the base and accelerate development strategies leading to sustained agricultural, industrial
The unlimited panorama of opportunities in the uses and applications of bio-technological developments has only just begun to emerge and conventional wisdom suggests that the panacea for increasing global food production and improvements in the quality of life has been found.
The majority of the developing world however lacks the financial resources to meet both capital and recurrent costs in exploiting such opportunities.
THE ISSUES IN PERSPECTIVE
The principal agricultural policy objectives of most of the developing countries of the world can be briefly summarized:
(i) Continuous and sustained increases in agricultural production and agricultural productivity under production systems which are environmentally friendly and sustainable,
(ii) Increase in opportunities in the rural sector,
(iii) Increase in household earnings and household savings from agricultural-sourced incomes,
(iv) Improvements in the nutritional and welfare status of these households.
(v) Increase in agricultural export earnings.
The economic and agricultural transformation processes which have already occurred in some developing countries and the status of agricultural research in other countries confirm significant opportunities for the future.
Most developed countries have been involved to various degrees in the b a s i c s c i e n c e s w h i c h g a v e r i s e t o b i o t e c h n o l o g y . S u c c e s s f u l commercialization of laboratory findings in the years ahead will offer many opportunities in agriculture, horticulture and forestry. Beneficial applications will also be found in the areas of environmental hygiene, pollution control and the recycling of wastes. These biotechnological developments will certainly impact upon third world economies in the following way:
(i) Induce changes in production systems, structures and costs.
(ii) Change demand/supply relationships of traditional inputs.
(iii) Substitution of traditional products with new products.
(iv) Increase the opportunities for agricultural pursuit.
(v) Impact on employment, income and consumption patterns.
(vi) Alter the existing market structures.
What the developments cannot guarantee is equity and efficiency in the distribution of benefits in order to maximize social welfare goals.
DEFINITION AND SCOPE Biotechnology
Very simply, biotechnology refers to any technique that uses living organisms to make or modify any products to improve plants or animals or to develop microorganisms for specific uses. These techniques include the use of new technologies such as recombinant DNA, cell fusion or other new processes.
The Overseas Development Institute (UK) (September 1988) defines the concept as follows:
"Biotechnology applies scientific and engineering skills, disciplines and principles to enable nationals to be processed by biological agents resulting in faster and more accurate breeding programmes for plants, animals and micro-organisms".
Of the several definitions available, it is evident that biotechnology integrates several recent advances in basic molecular biological research and encompasses many facets of management and manipulation of biological systems. In this regard, the very nature of biotechnology is related to several disciplines:
(i) In Natural Sciences -genetics, biotechnology, physiology and microbiology,
(ii) In Engineering - fermentation technology, production engineering, industrial chemistry, microbiology, etc.
B I O T E C H N O L O G Y IN SUGARCANE - OVERVIEW The principal objective of cane growing countries is to continuously increase production efficiencies at lower costs. This objective now seems mandatory given the present economics of world sugar production. By- products from sugarcane production and processing also lend themselves to tremendous value-added opportunities.
The production of non-sucrose sweeteners by biotechnological processes may impact negatively on the current glut of beet and cane sugar stocks on the world market. Sugar prices continue to be low because of the inflexibility of the industry to structurally adjust downwards in the short run given the nature of the cane growing cycle. Further, intensification of competition from fructose syrups, semi-synthetic sugars (aspartame) and artificial sweeteners such as acesulfame K continues unabated.
Modern biotechnology and related developments will impact on the sugarcane industry over the next 5-10 years in the following areas.
(i) Seed Material/Planting Material - New varieties of sugarcane plants carrying novel genetic traits such as pest or disease r e s i s t a n c e , i m p r o v e d y i e l d a n d q u a l i t y a t t r i b u t e s , n e w technologies, new production systems, etc.
(ii) Agricultural (Sugarcane) Microbiology - This will become a real p o s s i b i l i t y t h r o u g h t h e use o f g e n e t i c a l l y e n g i n e e r e d microorganisms as biological control agents for pests and diseases or as inoculants to stimulate plant growth and reduce fertilizer (iii) Sugarcane Diagnostics - Biotechnology will assist in the control of sugarcane diseases by providing rapid diagnosis on which to b a s e decisions on fungicide applications and other c o n t r o l measures. Further, results will assist in the identification of diseases of quarantine significance.
115 (iv) Research Programmes - These will develop insect and pest-
resistant commercial varieties, high-yielding commercial varieties, varieties with high sucrose content and varieties with other specific attributes, e.g. ratooning ability, minimum lodging, maturity times,
REVIEW OF BIOTECHNOLOGICAL D E V E L O P M E N T S IN T H E GENETIC IMPROVEMENT OF SUGARCANE Genetic improvement of sugarcane varieties in different countries has been taking place at varying levels of intensity and with different levels of resource commitment. Research programmes have been financed both by the public and private sector and are of critical importance in countries that depend on the export of sugar and related by-products to continuously generate export earnings.
T h e principal focus of such research p r o g r a m m e can be briefly summarized:
(i) To develop insect and pest-resistant commercial varieties (ii) To develop high-yielding commercial varieties (iii) To develop varieties with high sucrose content (iv) To develop varieties with other specific attributes, e.g. ratooning
ability, minimum lodging, maturity times, etc.
The principal difficulty with sugarcane breeding programmes emanates from the genetic concern that the sugarcane plant is octo or decaploid and is highly heterozygous. Given these conditions it normally takes about 10-15 years to commercialize a particular variety for selected traits. Any research technique which can circumvent these inherent difficulties will therefore provide enticing opportunities for the cane growing economies of the world.
Micro propagation of the sugarcane plant is possible from cultures of auxiliary buds and from calli. Such a technique enables the rapid propagation of virus free plants and can therefore shorten the quarantine period by two to three years.
On the other hand in-vitro micro propagation also offers pragmatic opportunities in effective storage facility requirements which are necessary for sugarcane propagation u n d e r traditional breeding programmes.
Within recent times, sugarcane researchers in Hawaii, Taiwan, Fiji, Cuba and Argentina have been successful in regenerating plants from calli obtained from stem or leaf cuttings. Similarly, and simultaneously, the IRAT Institute in France has developed a technique to inoculate pathogens into young plantlets regenerated from calli. Cuba has also made encouraging advances in this regard.
Protoplast fusion could also stimulate interest in the years ahead. Roque (1984) reported that it could be of great importance in the propagation of several varieties which are sterile. Work of this nature was initiated in 1981 at Piracicaba Campus, University of Sao Paulo, Brazil.
SOCIO ECONOMIC DIMENSIONS OF B I O T E C H N O L O G I C A L D E V E L O P M E N T
Generally, there appears to be some degree of concurrence among biological researchers, policy makers and planners that quantitative estimates of the likely impact of biotechnology on agriculture are difficult to obtain. T h e explanation resides in paucity of solid and reliable information to develop meaningful cost/benefit analyses.
Further, the reluctance of independent scientists, private farms and industrial concerns to divulge financial information c o m p o u n d s this p r o b l e m . N o t w i t h s t a n d i n g , there are p o s i t i v e s o c i o - e c o n o m i c benefits.
(i) Intensification of agricultural production across the globe.
(ii) Increasing and sustaining high levels of agricultural productivity, per unit land area of tropical and subtropical crops.
(iii) Bringing into the productive stream agricultural lands formerly classified as marginal.
(iv) Reduction in recurrent expenditures for production, e.g. reduction in cost of pesticides.
The negative aspects will be amplified in a later section.
Barker (1989) in his assessment of the potential economic impact of biotechnology in the third world explained and emphasized all the salient factors which can be briefly summarized as follows:
(i) Biotechnology will contribute modestly to increases in agricultural productivity in the area of crop production (1.5% - 2.2% per annum). He argues that yield plateaus have been achieved in some major crops and that even these modest increases will not be forthcoming in the absence of biotechnology.
(ii) Biotechnology is knowledge-intensive and often location-specific.
Biotechnology is potentially a "scale-neutral" technology but its application could contribute to serious, long term negative consequences for global trade and development.
S U M M A R Y OF ADVANTAGES A N D DISADVANTAGES TO DEVELOPING COUNTRIES
Advantages
One school of thought argues that early participation in new technologies is axiomatic for maintaining a leadership and thereby creating future wealth and employment. Atkinson & Mavituna (1983) have outlined three major factors in which the current interest in biotechnology and belief in its expansion are founded:
(i) Biotechnology can utilize raw materials obtained from renewable resources, i.e., cereal crops, celluloses and lignocelluloses.
(ii) Biotechnological processes appear to have advantages over the chemical processing of vegetable materials. Improvements in process technology can improve the efficiency of biotechnological industry.
(iii) A wide range of product appears possible both as a result of traditional and raw biological methods. Genetic engineering offers the promise of new products, increasing yields of existing products and modification of existing products.
Specific advantages clearly discernible for sugarcane are as follows:
(i) Rapid propagation of planting material.
(ii) High yielding cultivars will be produced at low cost, (iii) Planting material will be free from pathogens and contaminants, (iv) Expectation of increased agricultural productivity, (v) Value-added opportunities from fermentation technologies and
associated development of down stream industries.
Disadvantages
(i) T h e primary agricultural, agro-industrial and food processing sector may suffer commercialization of biotechnological products through substitution effects and changes in processes and products.
Multinational Corporations will continue to play a dynamic and technological leading role in these developments.
(ii) The existing trading pattern between the developed and developing world will be significantly altered to the d e t r i m e n t of the developing countries. Because new products will emerge primarily from the developed world and commercialization will make traditional production systems and processes obsolete. Undesirable effects will include unemployment, loss of income and possibly poverty and malnutrition. New production processes and systems may not produce sizeable economic benefits since they are more than likely to be capital intensive.
(iii) Effects on farming systems. Sugarcane production in most countries shows a dualistic production pattern - large commercial plantations side by side with small farmers. Biotechnological improvements in varieties with desirable agronomic traits will clearly s t r e n g t h e n the o p p o r t u n i t i e s for large c o m m e r c i a l plantations vis-a-vis small farmers. Large plantations have highly developed infrastructure, management and operational skills, investment in research and development facilities and stable organized marketing and financing arrangements. On the other hand, the small farmers lacking these will not be able to adopt the new technologies.
(iv) Reduction in genetic diversity. Special arrangements and facilities will be required to safeguard genetic material with desirable traits.
(v) S o m e c o u n t r i e s will not have t h e t e c h n i c a l , financial a n d institutional resources to optimize the benefits from the new technologies and may lag behind in the biotech revolution.