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Genetic Engineering of Cuban Rice Cultivars: Present and Perspectives
Plant Genetic Engineering: Towards the Third Millennium A.D. Arencibia (Editors) 2000 Elsevier Science B.V.
95
Genetic Engineering of Cuban Rice Cultivars: Present and Perspectives M Pujol*, Y Coil, J Alfonso,R Armas, C Hern&ndez,M P6rez, A Gonz&lez, E Aguiar Centro de Ingenieria Gen6tica y Biotecnologia de Sancti Spiritus, Apdo 83, Sancti Spiritus 60 200, Cuba.* Author for correspondence Introduction Rice is by far the most important crop in the developing world in terms of production and consumption and one of three most important crops all over the world. Just Indica-type rice varieties feed more than 2 billion people, predominantly in developing countries. This plant is well adapted to hot and humid environments, and is grown widely in both tropical and temperate regions. In Cuba, rice is the second crop in importance in cultivated area, just behind the sugarcane, although the domestic rice demand is largely unsatisfied by a local production of less than 300 000 TM. The per capita consumption of rice in Cuba is more than 40 Kg/year. Rice has been -and is nowadays- the basic source of calories in the Cuban people diet. It's usually eaten every day, and for most of the population it's common to have it twice a day. By-products from rice industrial processing are used in animal feeding and alternative sources of energy. Many constraints that use to limit rice production around the world are now being approached by genetic engineering. Among these, alien genes for resistance to insect pests is the one on which more effort have been dedicated, but also relevant are virus resistance, fungi resistance, herbicides resistance, and starch content modification. Some others, like abiotic stresses resistance, are not at the same research stage, but will be gaining importance as the basic studies involved in their under-standing become more advanced, providing the ways for more ap-plied investigations. Research on the manipulation of regulatory sequences dealing with the expression of the engineered genes just in desired plant tissues and only during required developmental periods of the plants have been reported in recent years, which will effectively help to avoid some of the present concerns on genetically engineered plants. These approaches also offer an alternative to the plant breeder who searches for genes very difficult or impossible of being bred by conventional genetics, because it often takes many years, if possible, for the incorporation of one of the mentioned genes into an agronomic important genetic background.
Features and Constraints of Rice Production in Cuba Most of the rice production in Cuba is obtained in large state-run enterprises where the crop is cultivated in two seasons. Cuban cultivars-Indica-type all of them obtained in a wide breeding program conducted from the beginning of the 60's by local breeders with the
96 assistance of IRRI (International Rice Research Institute) and other foreign experts are grown in these areas. Although many of these cultivars yield over 6.0 TM/ha in experimental conditions, the average yield in production areas hardly surpass 3.2 TM/ha at present, mainly because of the lack of funding and human resources needed for cultivation under high input conditions. Private sector -the vast majority composed of small-scale farmers- account for more than one third of the national rice production. These farmers cultivate rice in low input conditions, mainly during the rainy season. Among biological factors limiting rice production in Cuba, competition of weeds and red rice are of outstanding importance. Control of weeds occurring in rice growing areas demands a significant budget for chemical control in public enterprises. For the relevant part of the private sector that grows rice in rained culture and have no funding for chemicals, weed control by mechanical means suppose a big effort and a time consuming task. Fungal diseases are also an important limitation for rice production in Cuba. The blast disease caused by Pyricularia oryzae, the sheath blight caused by Rhizoctonia, and more recently the sheath rot caused by Sarocladium oryzae, are permanent threats for rice fields. Insect pests attacking rice in Cuba are leaded by the rice water weevil (RWW)
Lissorhoptrus brevirostris. Other pests of importance for rice are the stinkbug Oebalus insularis, the armyworm Spodoptera frugiperda, and the Homopteran Togasodes oryzicola. In addition, during the last few years the mite Steneotarsonemus spinki was found to be closely related to outbreaks of the sheath rot disease. Since about 10 % of the rice production take place in salt affected areas, salinity is one of the main abiotic factors limiting rice yields in Cuba. Droughts also significantly lower yields, mainly for farmers depending on rains for rice production. Occasional temperatures under 15~ occurring in Cuba during the winter, specially if they coincide with the anthesis stage, reduces pollen viability and can remarkably reduce yield. Hybrid technology for rice production has not been applied in Cuba because of lack of labor force. Goals and Results A few years ago, biotechnology research on rice in Cuba was restricted to some attempts for anther culture, and selection of mutants from somaclonal variation or irradiated calli. When at the Center for Genetic Engineering of Biotechnology of Sancti Spiritus we decided to start studying the potential of genetic engineering for approaching genetic improvement of local rice varieties, we found that almost no basic research on cell and tissue culture of this specie was done previously. Our primary goal was then to set up procedures for in vitro manipulation of the Cuban rice varieties. First, procedures for calli induction from mature seeds and maintenance, as well as for plant regeneration in the main local rice cultivars were established. From an initial 25 % of calli regenerating plants and about 1 average plant per callus obtained in the beginning, protocols were refined in such a way allowing some times 80 % of efficiency and more than 3 plants per callus, as reported in Coll et al. (1996). Additionally, we created conditions for induction of calli from rice coleoptiles as an alternative source of explants (Coll et al., 1997).
97 A very versatile regeneration medium for Cuban rice cultivars, including kinetin, BAP, and NAA in MS medium (Murashige and Skoog, 1968) was found to work efficiently for the cultivars in study (Coll, et al., 1998). Procedures for the isolation of physiologically active protoplasts from the Cuban cultivar Perla using etiolated leaves and roots were established. Transformation of protoplasts from both sources, as well as determination of transient GUS expression has been routinely performed in our laboratory. In parallel, studies to explore potential insecticidal molecules against two key pests of rice in Cuba have been performed in our laboratory. The digestive activity of the armyworm Spodoptera frugiperda was first characterized (Alfonso et al., 1996).
S. frugiperda a-amylase showed maximal activity at pH 8.5-9.5 in Tris buffer (Alfonso et al., 1997). S. frugiperda a-amylase activity in non-denaturing PAGE was observed during larval life, and no difference among the 5 different isoenzymes was detected. Proteinaceous inhibitors from wheat and barley were studied. The tetrameric inhibitor inhibits 25 and 60 % respectively of the total a-amylase activity at 7 and 11 days of larval life cycle. Inhibition with dimeric and monomeric inhibitors was not significant. Protease activity of the trypsin type in the larvae midgut was also detected. It was inhibited at 64 % by 5 gg of CMe proteins from barley cv. Bomi. We have also characterized the digestive protease activity of the rice water weevil as the first step for identifying proteins with inhibitory potential for plant protection strategies. Azocasein quantitative enzymatic assays (Table 1) and gelatinolytic activity on SDS-PAGE showed the existence of a complex proteolytic system in the RWW gut fluid, in which cysteine proteinase activity clearly predominates. Table 1._Responseof RWW digestive proteinases to various specific proteinase inhibitors. Inhibitors
E-64 (1) Kunitz trvosin inhibitor Leupeptin (2) Aprotinin PMSF (3) ED TA (4) Pepstatin A (5) Egg chickencystatin (6) 1+2+3+4+5+6
Concentration 280 ~M 100 ua/ml 200 ~M
Inhibition (%)a + SE 78.1 + 1.1 5 3 --I--(~ 1 b 75.3 + 1.8
ProteinaseSpecificit Y Cysteine Serine Cysteine/Serine
5.1 ~,M
6.9 + 0.8
Serine
2 pM
6.5 + 1.0
Serine
10 ~,M
1.5 + 0.5
Metallo
26.4 ~M
17.7 + 3.8
Aspartyl
10 tJg/ml
49.1 + 0.7
Cysteine
c
95.0 + 1.3
All
a : Data are expressed as percent of inhibition activity, calculated according to the formulae : (1 - SAi/SAc) x 100, where SAi represents the specific activity in presence of inhibitor, and SAc represents the control specific activity to which no inhibitor was added. Each datum is the mean of four determinations + SE. b: Activation instead of inhibition, c : The same concentration each as when individually used.
We concluded that the tetrameric and CMe trypsin inhibitors might be taken into account in genetic engineering strategies for protecting rice plants against S. frugiperda attack. Furthermore, several Bacillus thuringiensis strains were screened for toxicity against S.
98
frugiperda reared in artificial diet. A B. thuringiensis strain showing high toxicity was selected (Alfonso et al., 1994) and the gene coding for its 8-endotoxin was isolated and cloned in expression vectors. The recombinant 6-endotoxin protein expressed in Escherichia coli was compared with the natural protein in feeding experiments, demonstrating its toxic potential. Transformation of Cuban rice cultivars have been attempted both particle acceleration and
Agrobacterium tumefaciens. Although conditions were optimized for efficient transient expression in calli (Men6ndez, et al., 1999), no plants have been recovered from bombarded tissues. We identified a promoter driving strong expression of the GUS gene in bombarded immature rice embryos using the gene gun device.
However, Agrobacterium tumefaciens have proven to be an efficient vehicle to deliver foreign DNA into cells from Cuban rice cultivars. We have developed a procedure allowing so far recovering of transgenic rice plants from 3 cultivars. These plants, transformed with the GUS gene, were selected for hygromicin resistance. Molecular characterization of the transformed plants showed that most of the plants carry the introduced genes and express the active GUS protein. Transformation experiments aimed to introduce genes coding for antifungal proteins are in process. Chitinase, glucanase and PRs genes are combined in binary expression vectors in order to constitutively express antifungal proteins in transgenic rice plants from local cultivars. Transgenic rice plants will be tested for resistance to fungal diseases caused by Rhizoctonia, Pyricularia, and Sarocladium.
A Bacillus thuringiensis gene will be also introduced shortly into rice to study its insecticidal potential against the rice water weevil (Lissorhoptrus brevirostris) larvae. At present, several recombinant cysteine proteinase inhibitors are being evaluated to test their ability to interfere with the digestive activity from the RWW.
Future Prospects A strategy for the search of other Bacillus thuringiensis strains carrying novel 5-endotoxin genes was designed in our laboratory. Introduction of 8-endotoxin genes coding for insecticidal proteins differing in their RWW midgut receptor might both enhance efficiency of control and delay appearing of insect resistance. The introduction into rice of insecticidal genes with different mode of action, such as protease inhibitors or lectins, might also contribute to this purpose. Since some pests of economical importance for rice in Cuba are not of the chewing type, eg. the stinkbug Oebalus insularis, the Homopteran Togasodes oryzicola, and the mite Steneotarsonemus spinki, the potential of insecticidal molecules different from 5-endotoxins of B. thuringiensis need to be explored. Besides chitinase, glucanase and PRs genes, other antifungal genes are envisaged to be introduced into rice for ensuring a longer lasting effectiveness of transgenic plants in the field. Among these, genes coding for systemic acquired resistance (SAR) would be of outstanding importance. The development of expression systems for specific expression of the desired proteins in particular tissues, organs, cell compartments, and plant developmental stages will be necessary to cope with the expectations created around transgenic rice plants.
99 In particular, it would be very convenient to set up systems capable of diminish the risk of gene escaping through the pollen from transgenic plants to sexually compatible wild or relative rice species, and/or controlling the viability of potential undesired hybrids. These is a technical development specially appropriated when creating transgenic plants carrying genes and could confer competitive advantage if unintentionally bred into natural populations. Transformation of local rice cultivars with genes targeted to improve the defense of the plant against the main environmental stresses limiting rice yields in Cuba (cold, drought and salinity) would enable biotechnology to help breeders to complement rice breeding for attaining genetic goals not feasible using conventional techniques. The introduction of genes capable of simplifying the technology for production of hybrid seed or providing rice cultivars with the uniqueness of perpetuating the advantages of hybrids will means a breakthrough for rice production in Cuba.
References Alfonso J, Coil Y, Armas R, Pujol M, Ayala JL, de la Riva G, Selman-Houssein G. Identificaci6n de una cepa de Bacillus thuringiensis con alta capacidad insecticida frente a la palomilla del maiz Spodopterafrugiperda. Centro Agricola 1994; 1: 19-25. Alfonso J, Ortego F, Sfinchez R, Garcia G, Pujol M, Castafiera P, Salcedo G. Wheat and barley inhibitors active towards a-amylase and trypsin-like activities from Spodoptera frugiperda. Journal of Chemical Ecology 1997; 23(7): 1729-1741. Alfonso J, Sfinchez-Monge R, Garcfa-Casado G, Coll Y, Armas R, Pujol M, Salcedo G. Determination and in vitro inhibition of a-amylase and protease activity in Spodoptera frugiperda. Biotecnologia Aplicada, 1996; 13(4): 293. Coil Y, Gonz~ilez A, Alfonso J, Armas R, Pujol M. Callus induction from indica rice coleoptiles. International Rice Research Notes 1997; 22(2): 17, 1997. Coll Y, Pujol M, Castillo D, Gonz~ilez A, Alfonso J, Armas R. Improvement of Indica rice (Oryza sativa L.) in vitro regeneration efficiency from callus mediated by stress. Cereal Research Communications, 1998; 26(2): 153-160. Coll Y, Castillo D, Gonz/dez A, Alfonso J, Armas R, M. Pujol. Improvement of indica rice plant regeneration from callus through manipulation of culture conditions. Biotecnologia Aplicada 1996; 13(4): 298. Men6ndez E, Arrieta J, Coego A, Pujol M, Coll Y, Gonz~ilez S, Selman G. Optimizaci6n de las condiciones de bombardeo para transformaci6n gen6tica de arroz (Oryza sativa) variedad Amistad 82. Revista CENIC Ciencias BioI6gicas 1999; 30 (1): 7-11. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissues cultures. Physiol Plantarum 1962; 15: 473-497.
95
Genetic Engineering of Cuban Rice Cultivars: Present and Perspectives M Pujol*, Y Coil, J Alfonso,R Armas, C Hern&ndez,M P6rez, A Gonz&lez, E Aguiar Centro de Ingenieria Gen6tica y Biotecnologia de Sancti Spiritus, Apdo 83, Sancti Spiritus 60 200, Cuba.* Author for correspondence Introduction Rice is by far the most important crop in the developing world in terms of production and consumption and one of three most important crops all over the world. Just Indica-type rice varieties feed more than 2 billion people, predominantly in developing countries. This plant is well adapted to hot and humid environments, and is grown widely in both tropical and temperate regions. In Cuba, rice is the second crop in importance in cultivated area, just behind the sugarcane, although the domestic rice demand is largely unsatisfied by a local production of less than 300 000 TM. The per capita consumption of rice in Cuba is more than 40 Kg/year. Rice has been -and is nowadays- the basic source of calories in the Cuban people diet. It's usually eaten every day, and for most of the population it's common to have it twice a day. By-products from rice industrial processing are used in animal feeding and alternative sources of energy. Many constraints that use to limit rice production around the world are now being approached by genetic engineering. Among these, alien genes for resistance to insect pests is the one on which more effort have been dedicated, but also relevant are virus resistance, fungi resistance, herbicides resistance, and starch content modification. Some others, like abiotic stresses resistance, are not at the same research stage, but will be gaining importance as the basic studies involved in their under-standing become more advanced, providing the ways for more ap-plied investigations. Research on the manipulation of regulatory sequences dealing with the expression of the engineered genes just in desired plant tissues and only during required developmental periods of the plants have been reported in recent years, which will effectively help to avoid some of the present concerns on genetically engineered plants. These approaches also offer an alternative to the plant breeder who searches for genes very difficult or impossible of being bred by conventional genetics, because it often takes many years, if possible, for the incorporation of one of the mentioned genes into an agronomic important genetic background.
Features and Constraints of Rice Production in Cuba Most of the rice production in Cuba is obtained in large state-run enterprises where the crop is cultivated in two seasons. Cuban cultivars-Indica-type all of them obtained in a wide breeding program conducted from the beginning of the 60's by local breeders with the
96 assistance of IRRI (International Rice Research Institute) and other foreign experts are grown in these areas. Although many of these cultivars yield over 6.0 TM/ha in experimental conditions, the average yield in production areas hardly surpass 3.2 TM/ha at present, mainly because of the lack of funding and human resources needed for cultivation under high input conditions. Private sector -the vast majority composed of small-scale farmers- account for more than one third of the national rice production. These farmers cultivate rice in low input conditions, mainly during the rainy season. Among biological factors limiting rice production in Cuba, competition of weeds and red rice are of outstanding importance. Control of weeds occurring in rice growing areas demands a significant budget for chemical control in public enterprises. For the relevant part of the private sector that grows rice in rained culture and have no funding for chemicals, weed control by mechanical means suppose a big effort and a time consuming task. Fungal diseases are also an important limitation for rice production in Cuba. The blast disease caused by Pyricularia oryzae, the sheath blight caused by Rhizoctonia, and more recently the sheath rot caused by Sarocladium oryzae, are permanent threats for rice fields. Insect pests attacking rice in Cuba are leaded by the rice water weevil (RWW)
Lissorhoptrus brevirostris. Other pests of importance for rice are the stinkbug Oebalus insularis, the armyworm Spodoptera frugiperda, and the Homopteran Togasodes oryzicola. In addition, during the last few years the mite Steneotarsonemus spinki was found to be closely related to outbreaks of the sheath rot disease. Since about 10 % of the rice production take place in salt affected areas, salinity is one of the main abiotic factors limiting rice yields in Cuba. Droughts also significantly lower yields, mainly for farmers depending on rains for rice production. Occasional temperatures under 15~ occurring in Cuba during the winter, specially if they coincide with the anthesis stage, reduces pollen viability and can remarkably reduce yield. Hybrid technology for rice production has not been applied in Cuba because of lack of labor force. Goals and Results A few years ago, biotechnology research on rice in Cuba was restricted to some attempts for anther culture, and selection of mutants from somaclonal variation or irradiated calli. When at the Center for Genetic Engineering of Biotechnology of Sancti Spiritus we decided to start studying the potential of genetic engineering for approaching genetic improvement of local rice varieties, we found that almost no basic research on cell and tissue culture of this specie was done previously. Our primary goal was then to set up procedures for in vitro manipulation of the Cuban rice varieties. First, procedures for calli induction from mature seeds and maintenance, as well as for plant regeneration in the main local rice cultivars were established. From an initial 25 % of calli regenerating plants and about 1 average plant per callus obtained in the beginning, protocols were refined in such a way allowing some times 80 % of efficiency and more than 3 plants per callus, as reported in Coll et al. (1996). Additionally, we created conditions for induction of calli from rice coleoptiles as an alternative source of explants (Coll et al., 1997).
97 A very versatile regeneration medium for Cuban rice cultivars, including kinetin, BAP, and NAA in MS medium (Murashige and Skoog, 1968) was found to work efficiently for the cultivars in study (Coll, et al., 1998). Procedures for the isolation of physiologically active protoplasts from the Cuban cultivar Perla using etiolated leaves and roots were established. Transformation of protoplasts from both sources, as well as determination of transient GUS expression has been routinely performed in our laboratory. In parallel, studies to explore potential insecticidal molecules against two key pests of rice in Cuba have been performed in our laboratory. The digestive activity of the armyworm Spodoptera frugiperda was first characterized (Alfonso et al., 1996).
S. frugiperda a-amylase showed maximal activity at pH 8.5-9.5 in Tris buffer (Alfonso et al., 1997). S. frugiperda a-amylase activity in non-denaturing PAGE was observed during larval life, and no difference among the 5 different isoenzymes was detected. Proteinaceous inhibitors from wheat and barley were studied. The tetrameric inhibitor inhibits 25 and 60 % respectively of the total a-amylase activity at 7 and 11 days of larval life cycle. Inhibition with dimeric and monomeric inhibitors was not significant. Protease activity of the trypsin type in the larvae midgut was also detected. It was inhibited at 64 % by 5 gg of CMe proteins from barley cv. Bomi. We have also characterized the digestive protease activity of the rice water weevil as the first step for identifying proteins with inhibitory potential for plant protection strategies. Azocasein quantitative enzymatic assays (Table 1) and gelatinolytic activity on SDS-PAGE showed the existence of a complex proteolytic system in the RWW gut fluid, in which cysteine proteinase activity clearly predominates. Table 1._Responseof RWW digestive proteinases to various specific proteinase inhibitors. Inhibitors
E-64 (1) Kunitz trvosin inhibitor Leupeptin (2) Aprotinin PMSF (3) ED TA (4) Pepstatin A (5) Egg chickencystatin (6) 1+2+3+4+5+6
Concentration 280 ~M 100 ua/ml 200 ~M
Inhibition (%)a + SE 78.1 + 1.1 5 3 --I--(~ 1 b 75.3 + 1.8
ProteinaseSpecificit Y Cysteine Serine Cysteine/Serine
5.1 ~,M
6.9 + 0.8
Serine
2 pM
6.5 + 1.0
Serine
10 ~,M
1.5 + 0.5
Metallo
26.4 ~M
17.7 + 3.8
Aspartyl
10 tJg/ml
49.1 + 0.7
Cysteine
c
95.0 + 1.3
All
a : Data are expressed as percent of inhibition activity, calculated according to the formulae : (1 - SAi/SAc) x 100, where SAi represents the specific activity in presence of inhibitor, and SAc represents the control specific activity to which no inhibitor was added. Each datum is the mean of four determinations + SE. b: Activation instead of inhibition, c : The same concentration each as when individually used.
We concluded that the tetrameric and CMe trypsin inhibitors might be taken into account in genetic engineering strategies for protecting rice plants against S. frugiperda attack. Furthermore, several Bacillus thuringiensis strains were screened for toxicity against S.
98
frugiperda reared in artificial diet. A B. thuringiensis strain showing high toxicity was selected (Alfonso et al., 1994) and the gene coding for its 8-endotoxin was isolated and cloned in expression vectors. The recombinant 6-endotoxin protein expressed in Escherichia coli was compared with the natural protein in feeding experiments, demonstrating its toxic potential. Transformation of Cuban rice cultivars have been attempted both particle acceleration and
Agrobacterium tumefaciens. Although conditions were optimized for efficient transient expression in calli (Men6ndez, et al., 1999), no plants have been recovered from bombarded tissues. We identified a promoter driving strong expression of the GUS gene in bombarded immature rice embryos using the gene gun device.
However, Agrobacterium tumefaciens have proven to be an efficient vehicle to deliver foreign DNA into cells from Cuban rice cultivars. We have developed a procedure allowing so far recovering of transgenic rice plants from 3 cultivars. These plants, transformed with the GUS gene, were selected for hygromicin resistance. Molecular characterization of the transformed plants showed that most of the plants carry the introduced genes and express the active GUS protein. Transformation experiments aimed to introduce genes coding for antifungal proteins are in process. Chitinase, glucanase and PRs genes are combined in binary expression vectors in order to constitutively express antifungal proteins in transgenic rice plants from local cultivars. Transgenic rice plants will be tested for resistance to fungal diseases caused by Rhizoctonia, Pyricularia, and Sarocladium.
A Bacillus thuringiensis gene will be also introduced shortly into rice to study its insecticidal potential against the rice water weevil (Lissorhoptrus brevirostris) larvae. At present, several recombinant cysteine proteinase inhibitors are being evaluated to test their ability to interfere with the digestive activity from the RWW.
Future Prospects A strategy for the search of other Bacillus thuringiensis strains carrying novel 5-endotoxin genes was designed in our laboratory. Introduction of 8-endotoxin genes coding for insecticidal proteins differing in their RWW midgut receptor might both enhance efficiency of control and delay appearing of insect resistance. The introduction into rice of insecticidal genes with different mode of action, such as protease inhibitors or lectins, might also contribute to this purpose. Since some pests of economical importance for rice in Cuba are not of the chewing type, eg. the stinkbug Oebalus insularis, the Homopteran Togasodes oryzicola, and the mite Steneotarsonemus spinki, the potential of insecticidal molecules different from 5-endotoxins of B. thuringiensis need to be explored. Besides chitinase, glucanase and PRs genes, other antifungal genes are envisaged to be introduced into rice for ensuring a longer lasting effectiveness of transgenic plants in the field. Among these, genes coding for systemic acquired resistance (SAR) would be of outstanding importance. The development of expression systems for specific expression of the desired proteins in particular tissues, organs, cell compartments, and plant developmental stages will be necessary to cope with the expectations created around transgenic rice plants.
99 In particular, it would be very convenient to set up systems capable of diminish the risk of gene escaping through the pollen from transgenic plants to sexually compatible wild or relative rice species, and/or controlling the viability of potential undesired hybrids. These is a technical development specially appropriated when creating transgenic plants carrying genes and could confer competitive advantage if unintentionally bred into natural populations. Transformation of local rice cultivars with genes targeted to improve the defense of the plant against the main environmental stresses limiting rice yields in Cuba (cold, drought and salinity) would enable biotechnology to help breeders to complement rice breeding for attaining genetic goals not feasible using conventional techniques. The introduction of genes capable of simplifying the technology for production of hybrid seed or providing rice cultivars with the uniqueness of perpetuating the advantages of hybrids will means a breakthrough for rice production in Cuba.
References Alfonso J, Coil Y, Armas R, Pujol M, Ayala JL, de la Riva G, Selman-Houssein G. Identificaci6n de una cepa de Bacillus thuringiensis con alta capacidad insecticida frente a la palomilla del maiz Spodopterafrugiperda. Centro Agricola 1994; 1: 19-25. Alfonso J, Ortego F, Sfinchez R, Garcia G, Pujol M, Castafiera P, Salcedo G. Wheat and barley inhibitors active towards a-amylase and trypsin-like activities from Spodoptera frugiperda. Journal of Chemical Ecology 1997; 23(7): 1729-1741. Alfonso J, Sfinchez-Monge R, Garcfa-Casado G, Coll Y, Armas R, Pujol M, Salcedo G. Determination and in vitro inhibition of a-amylase and protease activity in Spodoptera frugiperda. Biotecnologia Aplicada, 1996; 13(4): 293. Coil Y, Gonz~ilez A, Alfonso J, Armas R, Pujol M. Callus induction from indica rice coleoptiles. International Rice Research Notes 1997; 22(2): 17, 1997. Coll Y, Pujol M, Castillo D, Gonz~ilez A, Alfonso J, Armas R. Improvement of Indica rice (Oryza sativa L.) in vitro regeneration efficiency from callus mediated by stress. Cereal Research Communications, 1998; 26(2): 153-160. Coll Y, Castillo D, Gonz/dez A, Alfonso J, Armas R, M. Pujol. Improvement of indica rice plant regeneration from callus through manipulation of culture conditions. Biotecnologia Aplicada 1996; 13(4): 298. Men6ndez E, Arrieta J, Coego A, Pujol M, Coll Y, Gonz~ilez S, Selman G. Optimizaci6n de las condiciones de bombardeo para transformaci6n gen6tica de arroz (Oryza sativa) variedad Amistad 82. Revista CENIC Ciencias BioI6gicas 1999; 30 (1): 7-11. Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissues cultures. Physiol Plantarum 1962; 15: 473-497.