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The Efficiency of Natural and Artificial Pollinators in Plantain (Musaspp. AAB group) Hybridization and Seed Production
Annals of Botany 80 : 693–695, 1997
SHORT COMMUNICATION
The Efficiency of Natural and Artificial Pollinators in Plantain (Musa spp. AAB group) Hybridization and Seed Production R O D O M I R O O R T I Z and J O N A T H A N H. C R O U C H Plantain and Banana Improement Program, International Institute of Tropical Agriculture, High Rainfall Station, PMB 008 Nchia-Eleme, Riers State, Nigeria* Received : 28 September 1996
Accepted : 30 June 1997
Plantain-derived tetraploid hybrids are routinely crossed in Musa breeding programmes with diploid Musa accessions for the efficient generation of putative triploid hybrid seed. However, natural open pollination of these same tetraploid hybrids also consistently generates viable seed. The mean germination rate of such open pollinated seed was observed to be higher than that of seed generated from artificial pollinations. This may suggest that tetraploid Musa hybrids played a much more important role in the evolution of triploid Musa landraces than previously considered. Moreover, the elite performance of certain hybrids generated through such open pollination offers possibilities of new Musa breeding paradigms. The inferences of these observations for Musa evolution and the implications for Musa breeding are discussed. # 1997 Annals of Botany Company Key words : Banana, hybrid seed, Musa, open pollination, plantain, polycross, synthetic.
INTRODUCTION Plantains (Musa spp. AAB group) are triploid (2n ¯ 33 chromosomes) starchy bananas, whose seedless parthenocarpic fruits are eaten cooked because they are unpalatable when raw. This crop, together with yams (Dioscorea spp.), provided the basis for the development of tropical African agriculture (Jurion and Henry, 1969). Most plantains are almost completely male sterile but some landraces set seed after hand pollination with haploid (n) pollen from diploid (2n ¯ 22) bananas (Vuylsteke, Swennen and Ortiz, 1993 c). Despite plaintain’s partial sterility (due to the uneven chromosome number and irregular meiosis), promising tetraploid hybrids (2n ¯ 44) (Vuylsteke, Swennen and Ortiz, 1993 b ; Vuylsteke et al., 1995), combining desired pest resistance, high yields and acceptable fruit taste for local consumers have been developed through ploidy manipulations (Ortiz, Ferris and Vuylsteke, 1995). This was achieved by interspecific artificial hybridization of African plantain landraces with wild or cultivated diploid Asian bananas. On average, diploid species and landraces have more pollen than the few tetraploid landraces (Dumpe and Ortiz, 1996). These tetraploid hybrids are fertile and may set seed after pollination. The problem of seeded fruit can be solved by reducing the chromosome number to 33, thereby restoring sterility. Secondary triploids are easily obtained from tetraploid-diploid crosses (Ortiz, 1997). This paper reports on the seed set obtained after natural * For correspondence at : The Royal Veterinary and Agricultural University, Department of Agricultural Sciences, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark. Fax 45 3528 3465, e-mail RO!KVL.DK
0305-7364}97}11069303 $25.00}0
open pollination and artificial hand pollination of tetraploid plantain-derived hybrids and its implications for the improvement of the plantain genome. MATERIALS AND METHODS A total of 371 artificially cross pollinated bunches (one per plant) of seven primary tetraploid plantain hybrids (TMPx) were bagged, hand pollinated with pollen from three plantain-derived diploid hybrids (TMP2x) and re-bagged (Vuylsteke and Ortiz, 1995) at Onne (south-eastern Nigeria) from January 1994 to December 1995. Female inflorescences of an additional 168 plants from the same seven TMPx genotypes in the same crossing block were left un-bagged to allow open pollination during the same period. The seven TMPx were 548-9, 1658-4, 2796-5, 4698-1, 6930-1, 7002-1 and 7152-2 (Vuylsteke et al., 1993 b). In Musa, several flower bracts replace leaves at the transition from the vegetative to the floral stage. The flower bracts enlarge and differentiate into female (pistillate) flowers and then male (staminate) flowers. The flowers are biseriately arranged often in clusters of 12 to 20 per node. The basal nodes of the inflorescence bear pistillate flowers whereas the upper nodes bear staminate flowers. When the ovaries of the pistillate flowers develop into fruits the staminate flowers are exposed because the bracts lift concomitantly with an increase in the inflorescence axis (Stover and Simmonds, 1987). Artificial hand pollination and seed extraction from all bunches was carried out as described previously (Ortiz and Vuylsteke, 1995). Seed quality was determined by the observation of well-formed seed embryos with the aid of a stereoscope. In itro germination of Musa seeds followed the protocol of Vuylsteke, Swennen and De Langhe (1990).
bo970488
# 1997 Annals of Botany Company
694
Ortiz and Crouch—Natural and Artificial Seed Set in Plantain Hybrids
Student’s t tests for unequal sample sizes were used for mean comparisons between open and hand pollinations. The statistical comparisons were performed after checking for homogeneity of variances with the F test at the 5 % probability level. All statistical analyses were done using MSTAT-C software (Anonymous, 1990). RESULTS AND DISCUSSION Tetraploid Musa hybrids in pollination blocks at Onne (a location in the humid forest zone of south-eastern Nigeria) have been observed to set seed without artificial hand pollination, i.e. by open pollination (Table 1). Bats, honeybees and birds are among the most important natural pollinators of Musa at Onne (Mutsaers, 1993). Although the number of open pollinated bunches (OPB) bearing seeded fruits was smaller than that obtained through artificial tetraploid-diploid crosses, the seed set was statistically similar (Table 2). The percentage of OPB varied from 17 to 92 %, depending on the maternal tetraploid hybrid genotype. Thus, it may be possible to select tetraploid parents which have the same frequency of bunches bearing seeded fruits due to natural open pollination as that obtained through artificial hand pollination (range : 30–88 %). Seed quality, as determined by the presence of a well formed embryo, was the same after open or artificial hand pollination (see Table 1). Germination in the soil was higher for the open pollinated seeds than for the hybrid seeds obtained from hand pollination. In contrast, in itro germination of seeds after embryo culture was the same for both types of seeds.
These observations may have important inferences for the evolution and domestication of triploid plantains. For example, tetraploid Musa hybrid cultivars, which evolved through sexual polyploidization as in other crops (Harlan and De Wet, 1975), may have been a progenitor of the original plantains [instead of only diploids, as believed (Simmonds, 1995)]. The putative parental tetraploid cultivars might have become rare (or even extinct) in plantain’s Asia-Pacific centre of domestication because they were less preferred by farmers. A combination of factors, such as seeded fruits and long cycling of tall plants, are likely to have made these tetraploids less attractive than their triploid offspring. Subsequently a few triploid cultivars, which were selected by farmers, were taken by travellers from IndonesiaMalaysia to Africa during pre-Columbian times (Simmonds, 1995). African plantains (currently in excess of 100 landraces) then evolved through somatic mutations and farmers’ selections during centuries of cultivation in the humid lowlands of West Africa (De Langhe, 1969) which then became the secondary centre of plantain diversification. The selection of TMPs 548-9 (Vuylsteke, Ortiz and Ferris, 1993 a) a ‘ synthetic hybrid ’ derived from the open pollination of the primary tetraploid hybrid TMPx 548-9 (Ortiz et al., 1995), shows the feasibility of utilizing open pollination for further improvement of the plantain genome. This hybrid was selected in an early evaluation trial at Onne, because it had a heavy bunch that bore many large fruits. To optimize this evolutionary breeding scheme, Ortiz (1997) proposed that fertile tetraploid and diploid parents should be selected, based on their specific combining ability, and
T 1. Total number of bunches examined bearing seeds (A), seeds produced (B) and treated for in vitro (C ) and soil (D) germination (Onne January 1994–December 1995 ) After hand pollination with pollen from Open pollination
TMP2x 2829-62
TMPx 1297-3
TMPx 1448-1
Tetraploid TMPx
A
B
C
D
A
B
C
D
A
B
C
A
B
C
548-9 1658-4 2796-5 4698-1 6930-1 7002-1 7152-2
3 12 10 4 4 7 14
24 691 1426 592 524 128 1406
24 148 645 492 304 128 794
— 543 781 100 220 — 612
5 15 5 — 12 30 18
39 794 255 — 1192 2261 1295
39 744 145 — 390 480 1211
— 50 40 — 615 656 247
3 14 17 14 20 8 32
8 168 1988 1213 2409 2398 5062
8 144 1347 684 1408 638 2040
2 4 12 12 3 6 1
178 89 2430 2669 196 228 9
178 88 863 711 196 228 9
T 2. Mean characteristics of seed generated by seen tetraploid hybrids following open pollination (OP) or artificial pollination (X ) with the diploid hybrid TMP2x 1297-3, TMP2x 1448-1 or TMP2x 2829-62 After hand pollination (X) with pollen from
Bunches with seed (%) Seeds per bunch Seeds with embryo (%) In itro germination (%) Soil germination (%)
OP
1297-3
1448-1
2829-62
X-mean
t-test
37³10 87³22 56³2 15³3 6³2
55³6 114³38 54³3 20³4
53³4 93³32 58³3 14³3
74³7 60³13 53³2 13³2 1³1
60³4 90³18 55³2 16³2
P ¯ 0±0127 P " 0±05 P " 0±05 P " 0±05 P ¯ 0±0639
Mean³s.e. Statistical t-test was between OP and X-mean.
Ortiz and Crouch—Natural and Artificial Seed Set in Plantain Hybrids used in controlled and isolated polycross nurseries in locations where natural pollinators of Musa are abundant. Most offspring derived from this polycross will be triploids as a consequence of the lower viability of pollen from tetraploid hybrids compared to that from diploid hybrids. In this way, large populations of secondary triploid hybrids may readily be obtained, thereby facilitating the diversification and decentralization of Musa breeding. Such diverse populations may then be screened for rare allelic combinations which would confer high yields and adaptation to specific environmental niches and post-harvest preferences. LITERATURE CITED Anonymous. 1990. MSTAT-C : A microcomputer program for the design, management, and analysis of agronomic research experiments. Michigan State University, East Lansing. De Langhe E. 1969. Bananas (Musa spp.). In : Ferwarda FP, Wit F, eds. Outlines of perennial crop breeding in the tropics, Misc. Papers 4, Agric. Univ. Wageningen, The Netherlands, 53–78. Dumpe B, Ortiz R. 1996. Apparent male fertility in Musa germplasm. HortScience 31 : 1019–1022. Harlan JR, de Wet JMJ. 1975. On O. Winge and a prayer : the origins of polyploidy. Botanical Reiew 41 : 1–14. Jurion F, Henry J. 1969. Can primitie farming be modernised ? Publ. Inst. Natl. Etude Agron. du Congo, hors series. Belgian Coop. and Development Office, Brussels. Mutsaers M. 1993. Natural pollination of banana and plantain at Onne. MusAfrica 2 : 2–3.
695
Ortiz R. 1997. Secondary polyploids, heterosis and evolutionary crop breeding for further improvement of the plantain and banana gneome. Theoretical and Applied Genetics 94 : 1113–1120. Ortiz R, Ferris RSB, Vuylsteke D. 1995. Banana and plantain breeding. In : Gowen S, ed. Bananas and plantains. London : Chapman and Hall, 110–146. Ortiz R, Vuylsteke D. 1995. Factors influencing seed set in triploid Musa spp. L. and production of euploid hybrids. Annals of Botany 75 : 151–155. Simmonds NW. 1995. Bananas Musa (Musaceae). In : Smartt J, Simmonds NW. Eolution of crop plants. Essex : Longman, 370–375. Stover RH, Simmonds NW. 1987. Bananas, 3rd edn. London : Longman Scientific and Technical. Vuylsteke D, Ortiz R. 1995. Plantain-derived diploid hybrids (TMP2x) with black sigatoka resistance. HortScience 30 : 147–149. Vuylsteke D, Ortiz R, Ferris S. 1993 a. Genetic and agronomic improvement for sustainable production of plantain and banana in sub-Saharan Africa. African Crop Science 1 : 1–8. Vuylsteke D, Ortiz R, Ferris RSB, Swennen R. 1995. PITA-9 : A black sigatoka resistant hybrid from the ‘ False Horn ’ plantain gene pool. HortScience 30 : 395–397. Vuylsteke D, Swennen R, De Langhe E. 1990. Tissue culture technology for the improvement of African plantains. In : Fullerton RA, Stover RH, eds. Sigatoka leaf spot diseases of bananas. Montpellier, France : INIBAP, 316–337. Vuylsteke D, Swennen R, Ortiz R. 1993 b. Registration of 14 improved Tropical Musa plantain hybrids with black sigatoka resistance. HortScience 28 : 957–959. Vuylsteke D, Swennen R, Ortiz R. 1993 c. Development and performance of black sigatoka-resistant tetraploid hybrids of plantain (Musa spp., AAB group). Euphytica 65 : 33–42.
SHORT COMMUNICATION
The Efficiency of Natural and Artificial Pollinators in Plantain (Musa spp. AAB group) Hybridization and Seed Production R O D O M I R O O R T I Z and J O N A T H A N H. C R O U C H Plantain and Banana Improement Program, International Institute of Tropical Agriculture, High Rainfall Station, PMB 008 Nchia-Eleme, Riers State, Nigeria* Received : 28 September 1996
Accepted : 30 June 1997
Plantain-derived tetraploid hybrids are routinely crossed in Musa breeding programmes with diploid Musa accessions for the efficient generation of putative triploid hybrid seed. However, natural open pollination of these same tetraploid hybrids also consistently generates viable seed. The mean germination rate of such open pollinated seed was observed to be higher than that of seed generated from artificial pollinations. This may suggest that tetraploid Musa hybrids played a much more important role in the evolution of triploid Musa landraces than previously considered. Moreover, the elite performance of certain hybrids generated through such open pollination offers possibilities of new Musa breeding paradigms. The inferences of these observations for Musa evolution and the implications for Musa breeding are discussed. # 1997 Annals of Botany Company Key words : Banana, hybrid seed, Musa, open pollination, plantain, polycross, synthetic.
INTRODUCTION Plantains (Musa spp. AAB group) are triploid (2n ¯ 33 chromosomes) starchy bananas, whose seedless parthenocarpic fruits are eaten cooked because they are unpalatable when raw. This crop, together with yams (Dioscorea spp.), provided the basis for the development of tropical African agriculture (Jurion and Henry, 1969). Most plantains are almost completely male sterile but some landraces set seed after hand pollination with haploid (n) pollen from diploid (2n ¯ 22) bananas (Vuylsteke, Swennen and Ortiz, 1993 c). Despite plaintain’s partial sterility (due to the uneven chromosome number and irregular meiosis), promising tetraploid hybrids (2n ¯ 44) (Vuylsteke, Swennen and Ortiz, 1993 b ; Vuylsteke et al., 1995), combining desired pest resistance, high yields and acceptable fruit taste for local consumers have been developed through ploidy manipulations (Ortiz, Ferris and Vuylsteke, 1995). This was achieved by interspecific artificial hybridization of African plantain landraces with wild or cultivated diploid Asian bananas. On average, diploid species and landraces have more pollen than the few tetraploid landraces (Dumpe and Ortiz, 1996). These tetraploid hybrids are fertile and may set seed after pollination. The problem of seeded fruit can be solved by reducing the chromosome number to 33, thereby restoring sterility. Secondary triploids are easily obtained from tetraploid-diploid crosses (Ortiz, 1997). This paper reports on the seed set obtained after natural * For correspondence at : The Royal Veterinary and Agricultural University, Department of Agricultural Sciences, 40 Thorvaldsensvej, DK-1871 Frederiksberg C, Copenhagen, Denmark. Fax 45 3528 3465, e-mail RO!KVL.DK
0305-7364}97}11069303 $25.00}0
open pollination and artificial hand pollination of tetraploid plantain-derived hybrids and its implications for the improvement of the plantain genome. MATERIALS AND METHODS A total of 371 artificially cross pollinated bunches (one per plant) of seven primary tetraploid plantain hybrids (TMPx) were bagged, hand pollinated with pollen from three plantain-derived diploid hybrids (TMP2x) and re-bagged (Vuylsteke and Ortiz, 1995) at Onne (south-eastern Nigeria) from January 1994 to December 1995. Female inflorescences of an additional 168 plants from the same seven TMPx genotypes in the same crossing block were left un-bagged to allow open pollination during the same period. The seven TMPx were 548-9, 1658-4, 2796-5, 4698-1, 6930-1, 7002-1 and 7152-2 (Vuylsteke et al., 1993 b). In Musa, several flower bracts replace leaves at the transition from the vegetative to the floral stage. The flower bracts enlarge and differentiate into female (pistillate) flowers and then male (staminate) flowers. The flowers are biseriately arranged often in clusters of 12 to 20 per node. The basal nodes of the inflorescence bear pistillate flowers whereas the upper nodes bear staminate flowers. When the ovaries of the pistillate flowers develop into fruits the staminate flowers are exposed because the bracts lift concomitantly with an increase in the inflorescence axis (Stover and Simmonds, 1987). Artificial hand pollination and seed extraction from all bunches was carried out as described previously (Ortiz and Vuylsteke, 1995). Seed quality was determined by the observation of well-formed seed embryos with the aid of a stereoscope. In itro germination of Musa seeds followed the protocol of Vuylsteke, Swennen and De Langhe (1990).
bo970488
# 1997 Annals of Botany Company
694
Ortiz and Crouch—Natural and Artificial Seed Set in Plantain Hybrids
Student’s t tests for unequal sample sizes were used for mean comparisons between open and hand pollinations. The statistical comparisons were performed after checking for homogeneity of variances with the F test at the 5 % probability level. All statistical analyses were done using MSTAT-C software (Anonymous, 1990). RESULTS AND DISCUSSION Tetraploid Musa hybrids in pollination blocks at Onne (a location in the humid forest zone of south-eastern Nigeria) have been observed to set seed without artificial hand pollination, i.e. by open pollination (Table 1). Bats, honeybees and birds are among the most important natural pollinators of Musa at Onne (Mutsaers, 1993). Although the number of open pollinated bunches (OPB) bearing seeded fruits was smaller than that obtained through artificial tetraploid-diploid crosses, the seed set was statistically similar (Table 2). The percentage of OPB varied from 17 to 92 %, depending on the maternal tetraploid hybrid genotype. Thus, it may be possible to select tetraploid parents which have the same frequency of bunches bearing seeded fruits due to natural open pollination as that obtained through artificial hand pollination (range : 30–88 %). Seed quality, as determined by the presence of a well formed embryo, was the same after open or artificial hand pollination (see Table 1). Germination in the soil was higher for the open pollinated seeds than for the hybrid seeds obtained from hand pollination. In contrast, in itro germination of seeds after embryo culture was the same for both types of seeds.
These observations may have important inferences for the evolution and domestication of triploid plantains. For example, tetraploid Musa hybrid cultivars, which evolved through sexual polyploidization as in other crops (Harlan and De Wet, 1975), may have been a progenitor of the original plantains [instead of only diploids, as believed (Simmonds, 1995)]. The putative parental tetraploid cultivars might have become rare (or even extinct) in plantain’s Asia-Pacific centre of domestication because they were less preferred by farmers. A combination of factors, such as seeded fruits and long cycling of tall plants, are likely to have made these tetraploids less attractive than their triploid offspring. Subsequently a few triploid cultivars, which were selected by farmers, were taken by travellers from IndonesiaMalaysia to Africa during pre-Columbian times (Simmonds, 1995). African plantains (currently in excess of 100 landraces) then evolved through somatic mutations and farmers’ selections during centuries of cultivation in the humid lowlands of West Africa (De Langhe, 1969) which then became the secondary centre of plantain diversification. The selection of TMPs 548-9 (Vuylsteke, Ortiz and Ferris, 1993 a) a ‘ synthetic hybrid ’ derived from the open pollination of the primary tetraploid hybrid TMPx 548-9 (Ortiz et al., 1995), shows the feasibility of utilizing open pollination for further improvement of the plantain genome. This hybrid was selected in an early evaluation trial at Onne, because it had a heavy bunch that bore many large fruits. To optimize this evolutionary breeding scheme, Ortiz (1997) proposed that fertile tetraploid and diploid parents should be selected, based on their specific combining ability, and
T 1. Total number of bunches examined bearing seeds (A), seeds produced (B) and treated for in vitro (C ) and soil (D) germination (Onne January 1994–December 1995 ) After hand pollination with pollen from Open pollination
TMP2x 2829-62
TMPx 1297-3
TMPx 1448-1
Tetraploid TMPx
A
B
C
D
A
B
C
D
A
B
C
A
B
C
548-9 1658-4 2796-5 4698-1 6930-1 7002-1 7152-2
3 12 10 4 4 7 14
24 691 1426 592 524 128 1406
24 148 645 492 304 128 794
— 543 781 100 220 — 612
5 15 5 — 12 30 18
39 794 255 — 1192 2261 1295
39 744 145 — 390 480 1211
— 50 40 — 615 656 247
3 14 17 14 20 8 32
8 168 1988 1213 2409 2398 5062
8 144 1347 684 1408 638 2040
2 4 12 12 3 6 1
178 89 2430 2669 196 228 9
178 88 863 711 196 228 9
T 2. Mean characteristics of seed generated by seen tetraploid hybrids following open pollination (OP) or artificial pollination (X ) with the diploid hybrid TMP2x 1297-3, TMP2x 1448-1 or TMP2x 2829-62 After hand pollination (X) with pollen from
Bunches with seed (%) Seeds per bunch Seeds with embryo (%) In itro germination (%) Soil germination (%)
OP
1297-3
1448-1
2829-62
X-mean
t-test
37³10 87³22 56³2 15³3 6³2
55³6 114³38 54³3 20³4
53³4 93³32 58³3 14³3
74³7 60³13 53³2 13³2 1³1
60³4 90³18 55³2 16³2
P ¯ 0±0127 P " 0±05 P " 0±05 P " 0±05 P ¯ 0±0639
Mean³s.e. Statistical t-test was between OP and X-mean.
Ortiz and Crouch—Natural and Artificial Seed Set in Plantain Hybrids used in controlled and isolated polycross nurseries in locations where natural pollinators of Musa are abundant. Most offspring derived from this polycross will be triploids as a consequence of the lower viability of pollen from tetraploid hybrids compared to that from diploid hybrids. In this way, large populations of secondary triploid hybrids may readily be obtained, thereby facilitating the diversification and decentralization of Musa breeding. Such diverse populations may then be screened for rare allelic combinations which would confer high yields and adaptation to specific environmental niches and post-harvest preferences. LITERATURE CITED Anonymous. 1990. MSTAT-C : A microcomputer program for the design, management, and analysis of agronomic research experiments. Michigan State University, East Lansing. De Langhe E. 1969. Bananas (Musa spp.). In : Ferwarda FP, Wit F, eds. Outlines of perennial crop breeding in the tropics, Misc. Papers 4, Agric. Univ. Wageningen, The Netherlands, 53–78. Dumpe B, Ortiz R. 1996. Apparent male fertility in Musa germplasm. HortScience 31 : 1019–1022. Harlan JR, de Wet JMJ. 1975. On O. Winge and a prayer : the origins of polyploidy. Botanical Reiew 41 : 1–14. Jurion F, Henry J. 1969. Can primitie farming be modernised ? Publ. Inst. Natl. Etude Agron. du Congo, hors series. Belgian Coop. and Development Office, Brussels. Mutsaers M. 1993. Natural pollination of banana and plantain at Onne. MusAfrica 2 : 2–3.
695
Ortiz R. 1997. Secondary polyploids, heterosis and evolutionary crop breeding for further improvement of the plantain and banana gneome. Theoretical and Applied Genetics 94 : 1113–1120. Ortiz R, Ferris RSB, Vuylsteke D. 1995. Banana and plantain breeding. In : Gowen S, ed. Bananas and plantains. London : Chapman and Hall, 110–146. Ortiz R, Vuylsteke D. 1995. Factors influencing seed set in triploid Musa spp. L. and production of euploid hybrids. Annals of Botany 75 : 151–155. Simmonds NW. 1995. Bananas Musa (Musaceae). In : Smartt J, Simmonds NW. Eolution of crop plants. Essex : Longman, 370–375. Stover RH, Simmonds NW. 1987. Bananas, 3rd edn. London : Longman Scientific and Technical. Vuylsteke D, Ortiz R. 1995. Plantain-derived diploid hybrids (TMP2x) with black sigatoka resistance. HortScience 30 : 147–149. Vuylsteke D, Ortiz R, Ferris S. 1993 a. Genetic and agronomic improvement for sustainable production of plantain and banana in sub-Saharan Africa. African Crop Science 1 : 1–8. Vuylsteke D, Ortiz R, Ferris RSB, Swennen R. 1995. PITA-9 : A black sigatoka resistant hybrid from the ‘ False Horn ’ plantain gene pool. HortScience 30 : 395–397. Vuylsteke D, Swennen R, De Langhe E. 1990. Tissue culture technology for the improvement of African plantains. In : Fullerton RA, Stover RH, eds. Sigatoka leaf spot diseases of bananas. Montpellier, France : INIBAP, 316–337. Vuylsteke D, Swennen R, Ortiz R. 1993 b. Registration of 14 improved Tropical Musa plantain hybrids with black sigatoka resistance. HortScience 28 : 957–959. Vuylsteke D, Swennen R, Ortiz R. 1993 c. Development and performance of black sigatoka-resistant tetraploid hybrids of plantain (Musa spp., AAB group). Euphytica 65 : 33–42.