4 S. Pac. J. Nat. Sci., (2002), Vol. 20, pp 4 – 8 Section A: Featured Article Cytogenetics and Reproductive Biology of some BELE (Abelmoschus manihot Linn., Medic Sub-Species manihot) Cultivars

Anand P. Tyagi Department of Biology, University of the South Pacific, Suva, FIJI. Email: [email protected]

Abstract

Four locally grown (from Fiji Islands) and three imported (from Papua New Guinea), a total of seven BELE (Abelmoschus manihot Linn., Medic sub-species manihot) cultivars were investigated for their cytogenetics and reproductive biology. Chromosome counting from flower buds and root tips showed that chromosome number in all the seven cultivars does not exceed 2n=66. Pollen viability in all the seven cultivars was high, exceeding 85% from pollen staining technique and 78% from pollen germination technique. All the seven Bele cultivars were fully self-compatible. Cross-compatibility among four local cultivars and three cultivars from Papua New Guinea was very high. However cross compatibility between local (Fijian) cultivars and cultivars from Papua New Guinea was partial indicating some degree of genetic difference between cultivars from two countries. This could be due to differences in compatibility alleles between cultivars from Fiji and Papua New Guinea. Procedures such as cutting the style shorter and placing pollen grains to enhance and affect fertilisation to get cross seed were suggested.

1 General Introduction Bele or Vauvau ni Viti or Aibika’s tender leaves are eaten as green vegetable after cooking. Bele is a very characters such as palatability and digestibility. However nutritious vegetable, which contains lots of minerals and very little information is available about cytogenetics and vitamins. In the present investigation seven cultivars, four reproductive biology of this very important vegetable crop. locals and three from Papua New Guinea were used to There are conflicting reports about chromosome numbers study flowering pattern, pollen viability and chromosome in this species. There are several references in the numbers. Knowledge of flowering pattern, pollen literature to chromosome counts in Abelmoschus manihot viability and chromosome numbers is very essential in giving 2n=60, 66 and 68, but there have been no confirmed breeding new varieties of any crop plant. This study counts in Bele or other forms of A. manihot, for example, would help plant breeders to develop new improved and chromosome number in Abelmoschus manihot subspecies more nutritious varieties of Bele for local consumption and manihot chromosome number reported were 2n=60 export. (Teshima 1933; Chizaki 1934). However, Skousted (1935) and Kamalova (1977) reported 2n chromosome number as 66, while Kuwada (1957a and 1974) reported a 2 Introduction chromosome number as 2n=68. In the single subspecies BELE or VAUVAU NI VITI or AIBIKA (Abelmoschus chromosome number cannot vary from 2n=60 – 2n=68. manihot Linn., Medik subspecies manihot formerly The taxonomy of Abelmoschus manihot has been the known as Hibiscus manihot Linn) was most probably subject of much debate. In particular, A. manihot indigenous in South Eastern Asia and presumably carried subspecies tetraphyllus (another Bele cultivar) is now into Pacific Islands by early voyagers (Smith 1981). Bele recognised by some as a separate species, on the grounds is mainly grown for its fleshy green lobed leaves which are that it appears to be at a different ploidy level (twice the used as vegetable (Preston,1998). BELE has been widely chromosome number) from A. manihot subspecies cultivated in Melanesia and sub species manihot is manihot. The basis of the separation of the subspecies doubtless a cultigen with glabrous stems derived from manihot and tetraphyllus is rather confusing and wild, more pilose and prickly plants (Smith 1981). Bele uncovering. Ugale et al (1976) reported chromosome leaves and branch tips are used as vegetable. Young numbers as 2n = 130 while Joshi and Hardas (1976) leaves and branch tips are very nutritious and rich in observed 2n = 138 in subspecies tetraphyllus. minerals and vitamins. These are cooked and eaten as Similarly self and cross compatibility studies were greens. At least four different cultivars (varieties) are confined to making interspecific and intergeneric crosses recognised in Fiji, and several more from Papua New (Gadwal et al. 1968; Kuwada, 1974; Hamon and Yapo, Guinea. Because of its importance as a highly nutritious 1986). Knowledge of self and cross compatibility is and palatable vegetable, a varietal improvement important for making intraspecific crosses to improve the programme is underway at Sigatoka Research Station productivity of cultivated varieties of this species by (Pacific Regional Agricultural Programme) funded by the combining desirable characters from two cultivars. European Union. A number of improved cultivars have Therefore main objectives of the present study were (i) to been developed and are being tested for their vegetable study the cytogenetics particularly chromosome number yield. (Young green leaves and branch tips), and quality and chromosome morphology and (ii) reproductive 5 biology including pollen fertility and self and cross and anthesis time. Pollination and fertilisation compatibility within and among different cultivars. observations were recorded on twenty flowers to determine these aspects of reproductive biology. Same ten selected 3 Material and Methods plants from each cultivar were also used to assess self and Four locally grown cultivars of Bele namely; Local A (L- cross compatibility among these cultivars. A) – strapped leaf shaped, Local B (L-B) – round leaf Ten flowers from each of the 10 plants in seven Bele shaped, Local C (L-C) – Pawpaw leaf shaped and Local D cultivars were used for self and cross-pollination. Crosses (L-D) – cotton leaf shaped were investigated. In addition and reciprocal crosses were made among different to these four locally grown cultivars, three introduced cultivars. For crossing, flower buds emasculation was cultivars viz.; LA-9, LA-16 and LA-45 from Papua New carried out before anthesis i.e. a day prior to the estimated Guinea (PNG) were also included in this study. All these date of stigma receptivity. Selfing and crossing was carried seven cultivars were being grown and maintained at out around 9.00 am. Self and cross-pollinated flowers Sigatoka Research Station. were collected after three hours interval each starting 12.00 For the mitotic chromosome count, stem cuttings from noon, 3.00 pm and 6.00 pm i.e. 3, 6 and 9 hours interval to these seven cultivars were grown in glass house and observe pollen germination on stigma and pollen tube research laboratory in Biology Department. Size 12" growth through stylar tissue. Another set of 10 self and 10 diameter plastic pots were used. Growing medium cross pollinated flowers from each plant, in each cultivar contained peat/moss so that growing roots could be was left intact and covered (using perforated plastic bags) collected easily. Indole Acetic Acid (IAA) was used to to enable seed set. Pollination and pollen tube growth was quicken the rooting process in the cuttings. Ten cutting observed and assessed as described by Dumas and Knox from each cultivar were used to collect root tips and to (1983), in which styles are cleared and stained with aniline prepare them for chromosome analysis. blue and then observed under UV light using incidence Root tips were prepared by treatment with 0.1% fluorescence optics. Pollination was classified as Colchicine for 3 hours as intact roots (with cuttings). compatible if pollen tubes were observed growing freely Growing root tips were cut and fixed in 3 parts absolute up to the base of style or reaching the ovule. alcohol: 1 part of glacial acetic acid for 24 hour at 4oC in a Pollen viability or fertility was tested using staining refrigerator. After that these were stored in 70% ethanol and germinating methods on artificial medium. For at 4ºC, until used for chromosome count. Root tips were staining methods pollen from freshly collected flowers macerated in 1N HCL for 10 minutes at 60oC in a water were put on a glass slide and stained with aceto-carmine bath. Macerated root tips were squashed in a drop of stain. Viable (fertile) pollen grains were stained deep red. Aceto-orcein stain on a clean glass slide, covered with While nonviable (sterile) pollen grains stained light red or glass cover slip, heated and pressed to spread the did not take any stain at all. Ten flowers were used from chromosomes. Prepared slides were observed under bright ten selected plants in each of the seven cultivars. Five field illumination (Tyagi et. al 1991). Fluorescent dye counts were made from each slide using five fields of view DAPI (4/, 6-diamidino-2-phenylindole 2HCL) was also under light microscope. At least 200 pollen grains were used to count mitotic chromosomes in root tips. To use counted under each field of view and averaged. DAPI the root tips were prepared by transferring them to Pollen viability was also tested using pollen citrate-phosphate buffer, pH 6 (3 changes over 24 hours), germination method on artificial medium. The medium was prepared using 1% bacteriological agar (GIBCO), dissected and teased apart in a drop of 1 µl/ml of stock 10% (W/V) sucrose and 0.01% boric acid (Morgan, 1990). solution of DAPI (1 mg/ml, Coleman and Goff, 1985). Pollen grains from ten flowers from each ten selected The roots were then squashed on a glass slide and plants in each cultivar were tested for germination. Pollen observed under UV light using incidence fluorescence grains were regarded as germinated when pollen tube optics. length was at least twice the pollen grain diameter (Polito For Meiotic chromosome count, 10 plants were and Luza 1988). At least four hundred pollen grains were selected and tagged from each cultivar. Flower buds in a counted in five visual fields for each plate prepared from a range of sizes were collected from each plant in all the single flower. Average pollen viability percentage was seven cultivars. Collected buds were pricked and calculated by counting germinating and total number of immediately fixed in 1:3 aceto-ethanol for 24 hours and pollen grains in each count. Thus in both cases staining then transferred to 70% ethanol for storage at 4oC. Buds method and germination method percentage was calculated were then stained in Snow’s (1948) alcoholic hydrochloric dividing the number of viable pollen in each case by total acid carmine for several days at room temperature. Excess number (viable + nonviable) of pollen grains counted in stain was rinsed in three changes of 70% ethanol (1 hour each cultivar. each). Anther contents from appropriate size buds were mounted on a glass slide in a drop of aceto-carmine (double-staining) smeared, heated and pressed to burst 4 Results open the pollen mother cells to differentiate the 4.1 Cytogenetics chromosomes from cytoplasmic granules (Tyagi et. al. 1991). The results of meiotic chromosome counting are presented For reproductive biology experiments, 10 selected in Table 1. plants from each cultivar were used to determine flowering

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Table 1 Meiotic chromosome counts at prophase i and metaphase i in seven bele cultivars

Cultivar/Accession Chromosome Count Univalents Bivalents Multivalents From PNG

LA9 Occasional 1 or 2 Regular 31-32 Occasional 1 or 2 LA16 Occasional 1 only Regular 32-33 Occasional 1 only LA45 Occasional 1 or 2 Regular 31-32 Occasional 1 or 2

Local – (From Fiji) A-Strapped leaf shaped (L-A) Occasional 1 or 2 Regular 31-32 Occasional 1 or 2 B-Round leaf shaped (L-B) Occasional 1 Regular 32-33 Occasional 1 only C-Pawpaw leaf shaped (L-C) Occasional 1 or 2 Regular 31-32 Occasional 1 only D-Cotton leaf shaped (L-D) Occasional 1 only Regular 31-32 Occasional 1 or 2

Several times 33 pairs (bivalents) were observed, however many times it looked that there were some univalents or Table 2 Pollen viability percentage (average) in seven bele multivalents. It was relatively easy to identify and cultivars using staining technique. differentiate between bivalents and multivalents but difficult to differentiate between bivalents and univalents Cultivars Pollen Viability Percentage because of very small size of chromosomes. Overall Range Mean + SE LA-9 85.7 – 89.3 86.5 + 2.58 meiotic chromosome counting confirms that there were LA-16 83.4 – 88.1 85.3 + 3.47 2n=66 chromosomes in all seven cultivars. LA-45 86.8 – 92.3 88.3 + 3.47 In mitotic chromosome counts, many times it was L-A 95.6 – 98.0 96.8 + 3.31 difficult to count the single chromosomes individually due L-B 92.3 – 95.8 93.2 + 3.04 to clustering of chromosomes together. In all Bele L-C 91.2 – 96.7 94.1 + 4.12 cultivars, however the chromosome count was between 62 L-D 88.3 – 93.4 91.4 + 3.22 and 66. Because the highest count in each cultivar did not exceed 66, it is therefore safe to conclude that the 2n Pollen Germination Technique (diploid) chromosome numbers in these cultivars was 66. Table 3 shows the pollen germination percentage on artificial medium in seven Bele cultivars. 4.2 Reproductive Biology 4.2.1 Flowering Time Table 3 Pollen germination percentage on artificial medium in All the seven Bele cultivars under study start flowering in seven Bele cultivars.

June and continue until October in Fijian climatic Pollen Germination Percentage condition. There is very little difference in size and shape Cultivar Range Mean + SE of flowers these Bele cultivars, however, the colour varied from cream white to dark pale with varying degrees of red- LA 9 75.8 – 81.3 78.2 + 3.56 maroon spotting inside lower parts of corolla. LA 16 73.6 – 79.2 75.8 + 3.02 LA 45 78.3 – 84.5 82.3 + 2.04 4.2.2 Anthesis L – A 81.6 – 86.4 84.4 + 3.82 L – B 86.9 – 89.1 88.0 + 2.12 Flower opening was normally between 8 and 10 a.m. L – C 82.7 – 84.9 83.7 + 3.19 Occasionally flower may open at noon or in the afternoon. L – D 85.2 – 91.6 87.5 + 3.76 This is uniform in seven cultivars studied in this project.

4.2.3 Pollination and Fertilisation 4.2.5 Selfing Soon after opening of the flowers, stigmas become Selfing results are presented in Table 4. It is evident from receptive. Mostly self-pollination is the rule. Any cross- these results that all the seven cultivars are fully self- pollination is always due to vectors such as honey-bees, compatible, and no self-incompatibility allelic system is wasps, beetles or some species of butterflies or moths. By operating in any of the seven cultivars. afternoon pollinated/fertilised flowers starts drooping and by evening accessory parts turned light pink and fall off or 4.2.6 Cross Compatibility remain attached in drooping form depending upon Some reciprocal differences were observed in crossing. particular cultivars’ characteristic features. Crossing among PNG and locally grown (Fijian) cultivars (separately) was more successful than between PNG and 4.2.4 Pollen Viability Fijian cultivars. Results of cross compatibility among all the seven cultivars are presented in Table 5 (see Staining Technique Appendix). Results clearly shows that the three strains Pollen viability percentage as obtained using staining from PNG namely; LA9, LA16 and LA45 were fully cross technique is shown in Table 2. compatible among themselves. It means that pollen 7 germination and pollen tube growth was unrestricted reach near base in nine hours. Local Fijian cultivars were through stylar tissue and reached near the ovule within also fully cross compatible among themselves and pollen nine hours. However cross compatibility of PNG cultivars tube was observed reaching ovule within 9 hours after with Local Fijian Cultivars; L-A, L-B, L-C and L-D was pollination. partial. The pollen germination was delayed and pollen tube growth through stylar tissue was slow and could not

Table 4 Self -compatibility status in seven cultivars of bele.

Status of Pollen tube germination through styler tissue Cultivar 3 hours 6 hours 9 hours LA 9 Almost halfway through Near the bottom of style. Reached near the ovule. LA 16 Halfway through More than three fourth Reached near the base of style. through the style. LA 45 Halfway through do Do L – A Halfway through Three fourth way through. Almost reached ovule. The stylar tissue. L – B Halfway through do Do L – C Halfway through do Do L – D More than halfway through. Near the base of the style Reached ovule in all cases.

5 Discussion cultivars from PNG and locally grown Fijian cultivars. Tyagi and Singh (1998) reported high degree of self- 5. 1 Cytogenetics compatibility in self-pollinating mangrove species found in The size of chromosome in the family Malvaceae, as a Fiji. All three PNG cultivars were fully cross compatible whole, is very small, which makes it very difficult to count among themselves but only partially cross compatible with them properly. Previous reports on chromosome numbers four local cultivars. This could be due to some allelic varies from 2n = 60 – 2n = 68 (Teshima 1933, Chizaki differences in cultivar from two different places (Stebbins 1934, Skousted 1935, Kamalova 1977, Kuwada 1957 and 1958). 1974). Meiotic and mitotic chromosome number were It is well known for many species in the family observed in most cases as 2n = 66. Due to very small size Malvaceae cross incompatibility alleles are present and of chromosomes, sometimes it was difficult to identify operative reducing the chances of cross-pollination. individual chromosomes from cytoplasmic granules. That However, there are ways to overcome such barriers using was one reason why there were conflicting reports on certain other methods for cross fertilisation including chromosome counts in this species (Teshima 1933, cutting the style shorter and placing pollen grains to Chizaki 1934, Skousted 1935, Kamalova 1977, Kuwada enhance fertilisation to get cross seed. All the four local 1957 and 1974, Kamalova 1977). This information on cultivars were fully cross compatible among themselves. chromosome number in this species particularly for these Intra-specific (inter-varietal) hybrids thus can be obtained seven Fijian grown Bele cultivars would be useful for with character combinations from two different and further improvement of these cultivars by making intra- diverse cultivars (Preston 1998). specific crosses.

5. 2 Reproductive Biology 6 Acknowledgement Flowering in all seven cultivars in this species is spread Author wishes to thank the University of the South Pacific over 5-6 months and flowering normally occurs during Research Committee for supporting this investigation early hours of morning after the sunrise. This information through Grant No. 6292-1311-70766-15. will help breeder to plan selfing and crossing limiting to that time period and duration. References Pollen viability experiments confirmed very high Chazaki, Y. 1934. Breeding of a new interspecific hybrid pollen fertility in all seven cultivars of this species. Two between Hibiscus esculentus L., and H. manihot L. methods gave a good assessment of pollen fertility. Proc. crop Sci. Soc. Japan 6: 164-172. Cultivar differences in pollen viability were minimum and Coleman, A.W. and Goff, L.J. 1985. Application of no significant differences were observed in cultivars from fluorochromosomes to pollen biology. Mithromysin Papua New Guinea and locally grown cultivars from Fiji. and 4,6-Diamidine-2phenylindole (DAPI) as vital This is quite normal for well-established plant species to stains for quantitation of nuclear DNA, Stain have high pollen fertility (Tyagi et. al. 1995). technology 60: 145-154. Self and cross compatibility experiments brought it out Dumas, E. and Knox, R.B. 1983. Callose and clearly that all seven cultivars studied in this species were determination of pistil viability and incompatability. fully self fertile and hence self-compatible. The self Theoretical and Applied Genetics 67, 1-10. pollinating nature of the species is the reason for high self- Gadwal, V.R., Joshi, A.B. and Iyer, R.D. 1968. compatibility in all the seven cultivars. However the Interspecific hybrid in Abelmoschus through ovule and differences in cross compatibility were observed between 8

embryo culture. Indian J. Genet. Plant Breed. 28: Preston, S.R. 1998. Aibika/Bele – Abelmoschus manihot 269-274. (L) Medik. International Plant Genetic Resources Hamon, S. and Yapo, A. 1986. Perturbation induced within Institute (IPGRI) Publication. pp 97. the genus Abelmoschus by the discovery of a second Skousted, A. 1935. Chromosome number in the family edible okra species in West Africa. In: Maesen, L.J.G. Malvceae, J. Genet. 31: 263-296. Van Der, First International Symposium on Taxonomy Snow, R. 1963. Alcoholic hydrochloric acid-carmine as a of cultivated plants. Acta Hort. 182: 133-144. stain for chromosomes in squash preparations. Stain Kamalova, G.V., 1977. Cytological studies of some Technology. 38: 9-13. species of the Malvaceae. Uzbekistan Biologija Stebbins, G.L., Jr. 1958. The inviability, weakness and Zurnali 3: 66-69.8. sterility of inter-specific hybrids. Advances in Kuwada, H. 1957. Crossability in the reciprocal crosses Genetics, 147-215. between Abelmoschus esculentus and A. manihot, and Teshima, T. 1933. Genetical and cytological studies in an the characters and meiosis in F1 hybrids, Jap J. Breed. interspecific hybrid of Hibiscus esculentus and H. 7: 93-102. manihot. J. Fac. Agric. Hokkaido Univ. 4: 1-155. Kuwada, H. 1974. F1 Hybrids of Abelmoschus Tyagi, A.P., McComb, J. and Considine J. 1991. tuberculantus, and A. manihot with reference to the Cytogenetics and pollination studies in the genus genome relationship. Jap. J. Breed. 24: 207-210. Verticordia D.C. Australian Journal of Botany. 39: Morgan, A. 1990. The Breeding Biology of Verticordia 261-272. Section Catocalypta Benth. B.Sc., (Hons.) Thesis, Tyagi, A.P., Dass, C.R., Nathan, S., Racule, T. and Murdoch University, W.A. Australia. Lakhan, S. 1995. Pollen fertility status in some exotic Polito, V.S. and Luza, J.G. 1988. Longevity of pistachio flora of Fiji. South Pac. J. Nat. Sci. 14: 211-222. pollen determined in vitro germination. Journal of the Tyagi, A.P., and Singh V.V. 1998. Pollen fertility and American Society of Horticultural Science. 113, 214- intraspecific and interspecific compatibility in 217. mangroves of Fiji. Sexual Plant Reproduction. 11: 60-63.

Appendix

Table 5 Cross compatibility status of seven cultivars of bele (based on pollen tube growth after 9-hours).

Cultivars LA 9 LA 16 LA 45 L-A L-B L-C L-D

LA 9 Fully cross Fully cross Partially cross Partially cross Partially cross Partially cross compatible compatible compatible compatible compatible compatible (100%) (100%) (>50%) (>50%) (>50%) (>50%) LA 16 Fully cross Fully cross Partially cross Partially cross Partially cross Partially cross compatible compatible compatible compatible compatible compatible (100%) (100%) (>50%) (>50%) (>50%) (>50%) LA 45 Fully cross Fully cross Partially cross Partially cross Partially cross Partially cross compatible compatible compatible compatible compatible compatible (100%) (100%) (>50%) (>50%) (>50%) (>50%) L – A Partially cross Partially cross Partially cross Fully cross Fully cross Fully cross compatible compatible compatible compatible compatible compatible (>50%) (>50%) (>50%) (100%) (100%) (100%) L – B Partially cross Partially cross Partially cross Fully cross Fully cross Fully cross compatible compatible compatible compatible compatible compatible (>50%) (>50%) (>50%) (100%) (100%) (100%) L-C Partially cross Partially cross Partially cross Fully cross Fully cross Fully cross compatible compatible compatible compatible compatible compatible (>50%) (>50%) (>50%) (100%) (100%) (100%) L-D Partially cross Partially cross Partially cross Fully cross Fully cross Fully cross compatible compatible compatible compatible compatible compatible (>50%) (>50%) (>50%) (100%) (100%) (100%)

Above diagonal: crosses. Below diagonal: reciprocal crosses.