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Pollen Fertility and Breeding Biology of Three Species of Genus Ixora

A.P. Tyagi, T. Racule and P. Lal Department ofBiology, School of Pure and Applied Sciences The University of the south Pacific, P.OBox 1168, SUVA, Fiji [email protected]

ABSTRACT

Pollen viability (fertility) and breeding biology of three species of genus Ixora, namely; 1. chinensis, 1. javanica and 1. macrothyrsa were studied. Pollen germination in vitro showed that all three species had a very high pollen fertility (> 80%). Sone of the three species was fully selj-compatible. Only partial selj-compatibility was observed in 1. chinensis and 1. j avanica. Interspecific crossing results indicated cross incompatibility among the three species studied. Causes of cross incompatibility may include cryptic chromosomal differences, chromosomal aberrations and difference in chromosome numbers. Ways to achieve cross compatibility suggested to obtain hybrids among these species for further improvement

INTRODUCTION representative healthy plants were selected from each species. To determine pollen fertility, pollen was collected Genus Ixora belongs to family Rubiaceae, native to India, from freshly opened flowers. Viability (indicative offertility contains many species with small but very colour-full and status) of freshly collected pollen was assessed using a semi­ showy flowers. It is an important ornamental plant genus solid medium of 1% agar (bacteriological agar Gibco), 15% and used in home gardens, landscaping and as a:pot plant. w/v sucrose and 0.01% w/v boric acid. Agar was dissolved Various species of genus Ixora ranges from small trees to by heating and sterilized in an autocllwe, then poured into medium to small size shrubs. The three species studied are sterile 85-mm diameter Petri dishes to gel. Freshly collected all small size shrubs. These species bear bright red to cream pollen was dusted on to germination media and spread over colour flowers. Within the same species flower size and using a sterile glass rod. Petri-dishes were sealed and colour may vary depending upon variety and environmental placed iIi dark at 22 ±IOC for 24 hours (Tyagi et al., 1992). conditions. All the three species have linear to oblong leaves Pollen grains were scored as germinated when the pollen and an inflorescence terminal to axillary. Flowers are about tube length was at least twice the pollen grain diameter an inch long and born in clusters or crowded. Flowers are (polito and Luza, 1988). At least 500 pollen grains were mildly scented and very decorative (parham, 1972). Moths counted in each replication for calculation of percentage and butterflies (Lepidoptera) mainly pollinate flowers of pollen viability. these species. Plant Breeders are interested in breeding better and improved varieties of a large number of To study reproductive biology, i.e. self and cross ornamental plants. This work is still being persued with compatibility, number of self pollination and cross zeal and vigour all over the world. To develop improved pollination experiments were carried out. For self varieties of any ornamental plant it is important to know compatibility experiment, about fifty flower buds in an pollen fertility and reproductive biology of those plant inflorescence, were bagged using perforated plastic bags in species. Three species of the genus Ixora namely; Ixora five plants in each of the three species. To assess cross chinensis, Ixora javanica and Ixora macrothyrsa were compatibility, at least twenty flower buds which were about selected to study their pollen fertility and reproductive to open by next day, in five plants each of the three species biology during summer of 1995. were emasculated and bagged to avoid cross pollination by wind or vectors. Cross pollination was performed next day by collecting pollen from a desired male parent. Pollen were transferred to the stigmas of emasculated flowers using MATERIALS AND METHODS a fine camel hair brush or directly touching the burst anthers (from desired male parent) on to the stigma of emasculated flowers. Pistils were collected after 24, 48 and Three species of genus Ixora selected for pollen fertility and 96 hours in each of the self pollination and cross pollination reproductive biology study are Ixora chinensis (Linn.), Ixora experiments. Pistils were fixed in 1:3 acetoethanol using javanica (Linn.) and Ixora macrothyrsa (A.c. Smith). Five 100% glacial acetic acid and 95% ethyl alcohol. After 24

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hours pistils were transferred to 70% ethanol and stored in a on microscope glass slides in a drop or two of aniline blue refrigerator until used to observe pollen tube growth and covered with coverslips. The slides were then observed, through stylar tissue. Pistils were prepared for examination to determine pollen tube growth, under a fluorescence by transferring them to 10% w/v aqueous sodium sulphite microscope using ultraviolet filters. Callose in the pollen solution and autoclaving at 120 kpa at 121°C for 30 tube fluoresced bright yellow to yellow green, while the minutes. After autoclaving, pistils were stained for two to background tissue fluoresced pale-gray or blue (Martin, 24 hours in aqueous aniline blue solution containing 1959; Smith and McCully, 1978). 0.005% aniline blue and O.IM potassium dihydrogen orthophosphate. Mecerated (~ftened) pistils were squashed

RESULTS

Range and pollen viability percentage in three species of Ixora is presented in Table 1.

Table 1: Mean Pollen Viability (Fertility Status) percentage in three species of Ixora (average of five hundred pollen gtains).

Pollen Viability ~cies Rane:e 0/0 Mean (%) +S.E. Ixora chinensis 79.6 - 87.5 85.7:t 3.4 Ixora javanica 81.3 - 89.8 87.3 ± 2.9 Ixora macrothyrsa 76.5 - 85.2 82.8 ±3.3

Pollen fertility percentage was quite high (>80%) in all the union was observed. In two other species l javanica and l three species. However, Ixora javanica showed highest macrothyrsa pollen germination was observed in the former pollen germination percentage followed by Ixora chinensis while no germination was observed in the later species. and Ixora macrothyrsa in that order. Pollen tube growth in case of Ixora javanica was almost 113 of the stylar tissue after 48 hours and only 3/4 through stylar Results of self-pollination on pollen tube growth in three tissue after 96 hours (Table 2). In Ixora macrothyrsa pollen species of Ixora are shown in Table 2. Self-pollination tube could penetrate only halfway through stylar tissue after experimental results indicate at least partial self 96 hours indicating that this species is almost fully self­ incompatibility. In l chinensis pollen tube could reach the incompatible. ovule only after 96 hours and no male gamete discharge or

Table 2: Self pollination and pollen tube growth results of three species of Ixora.

Time of collection Species after pollination Observations

24 hours Pollen tubes just started to grow. Ixora chinensis 48 hours Pollen tubes penetrated halfway through stylar tissue. 96 hours Pollen tubes reached near the ovule.

24 hours Pollen germination was observed. Ixora javanica 48 hours Pollen tubes penetrated almost one third through stylar tissue. 96 hours Pollen tubes growth almost halfway through stylar tissue. 24 hours No pollen grain germinated. Ixora macrothyrsa 48 hours Pollen tubes penetrated almost one fourth through stylar tissue 96 hours Pollen tubes reached almost half way through stylar tissue. 9

In crosses and reciprocal crosses between I. chinensis and I. line blue species. Similarly complete cross incompatibility was j avanica partial self compatibility was observed. Crosses )bserved, recorded between I javanica and I macrothyrsa. In these and reciprocal .crosses between I. macrothyrsa and I. lJ"escence crosses, pollen germination (on stagmetic surface) was chinensis showed only pollen germination. Further pollen Ie pollen observed only 24 to 48 hours after pollination and pollen tube growth was restricted due to swollen pollen tubes tips mile the tubes could not penetrate the stylar tissue at all. indicating complete cross incompatibility between these two (Martin, Table 3: Cross compatibility status among three species of genus lxora.

Crosses / Reciprocal Time of collection Observations Crosses after pollination Ixora chinensis X 24 hours No Pollen germination was observed Ixora javanica 48 hours Pollen germinated, but no penetration through stylar tissue 96 hours Penetration occurred but no further growth through stylar tissue Ixora javanica X 24 hours No pollen germination :I pollen Ixora chinensis 48 hours Pollen germinated 96 hours Pollen tubes growth was restricted at the top of stylar tissue. Ixora chinensis x' 24 hours Pollen germination occurred Ixora macrothyrsa 48 hours Pollen tubes penetration observed 96 hours Swollen pollen tube tip - no further growth Ixora. macrothyrsa X 24 hours Pollen germinated Ixora chinensis 48 hours Pollen tubes penetrated through stylar tissue 96 hours Pollen tubes growth was blocked due to swollen end. Ixora Javanica X 24 hours No Pollen germination Ixora macrothyrsa 48 hours No Pollen germination 96 hours Pollen germinated but no pollen tubes penetration IJ and I Ixora macrothyrsa X 24 hours No pollen germination former Ixora javanica 48 hours Pollen germinated species. 96 hours Pollen tubes did not penetrate the stylar tissue lOst 1/3 h stylar rpollen lIe after DISCUSSION flower at the time when pollen was shed by anthers in Iy self- flowers (Bidwell, 1979). In some species, incompatible Pollen germination resuJts on artificial medium showed very pollen germinates and pollen tube grows down the style (as high pollen fertility (> 80%) in all three species. High in Ixora chinensis and I javanica) but the growth of the pollen fertility is a common feature in almost all tropical pollen tube is so slow that it rarely reaches the ovary in time and subtropical species (Tyagi et al., 1995; Tyagi and Singh to effect fertilization. The rate of growth of incompatible 1998). Minor variation observed in pollen viability pollen tubes can be influenced ~y the background genotype percentage could be due to genetic differences among three in which the major gene(s) governing the incompatibility species. reaction operates (Lewis 1954). Self incompatibility is an out breeding mechanism which maintains a high degree of Self pollination results among three species showed that all heterozygosity in a species (Grant, 1958; Simmonds, 1962). three species were partial to complete self-incompatible. Self incooipatibility mechanisms vary from one species to Self incompatibility is not uncommon in plant species. the others. These are either gametophytic or sporophytic Darlington and Mather, as early as 1949, stated that almost but one plant family apparently has one type only half of the species of flowering plants (excluding self­ (Williams, 1951). Incompatibility reactions are widespread. fertilizing species) have incompatible systems. Brewbaker, ­ such reactions can only be discovered by self pollination (1957) reported that a large number of species in 66 families experiments in controlled conditions. Differences between of flowering plants representing every major phylogenetic the two self-incompatibility systems have important line of the angiosperm, have one kind or the other self­ consequences for the plant breeder. These systems incompatibility systems. Self incompatibility may also determine how self-incompatibility can be used to breeder's result from timing differences in pollen and stigma advantage. Self-incompatibility, whether gametophytic or development such that conditions (nutritional or hormonal) sporophytic, presents two contrasting prospects to the plant are not favourable for pollen germination on the stigma of a breeder. On one hand" it wiU ,frustrate efforts to produce

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homozygous (inbreed) lines, but on the other it provides a Brewbaker, JL (1957): Pollen cytology and self­ way to hybridize two clones without emasculation and incompatibility systems in plants. l Hered., 48: 27-277. without resorting to genetic or cytoplasmic male sterility. Darlington, CD and Mather, K (1949): The Elements of Cross pollination results showed that none of the crosses Genetics. George Allen and Urwin, London. and reciprocal crosses attempted among three species were successful. This clearly indicated that all the three species Grant, V (1958): The regulation of recombination in plants. were cross-incompatible. Cross incompatibility among Symp. on Quant. BioI., 23: 337-363. related species could be due to several factors such as (i) incompatible poUen reaction in stylar tissue (ii) cryptic Grant, V (1975): Genetics of Flowering Plants. Columbia chromosomal differences and (iii) differences in University Press, London, pp.303-342. chromosome numbers. In present study it could be due to any or all of these factors (Grant, 1975). Populations evolve Lewis, Dc (1954): Comparative incompatibility in and become two separate species due to reproductive angiosperms and fungi. Adv. . Gene., 6: 235-285. barriers. Reproductive barriers arise due to accumulation of cryptic gene differences and chromosomal abbreviations Martin, FW (1959): Staining and observing pollen tubes in including chromosome numbers. This could be with or the style by means of fluorescence. Stain Technology, 34: without prior geographical isolation of two sub-populations. 125-128. Implications of these results are quite important to breeders. Parham, JW (1972): Plants of The Fiji Islands. Published by Various methods to achieve interspecific cross compatibility Authority of the Government Printer, Suva. may be tried such as application of weak sugar solution on to the surface of stigma just before hand pollination, cutting Polito, VS and Luza, JG (1988): Longivity of Pistachio and reducing the length of style. In cases where fertilization pollen determined by in vitro germination. lAm. Soc. Hort. has occurred but embryo abort due to incompatibility, Sci., 113: 214-217. embryos may be rescued and reared on artificial medium (Tyagi et ai., 1991). In this way new hybrid combinations Simmonds, NW (1962): Variability in crop plants: its uses may be obtained. However, cross compatibility may be and conservation. BioI. Rev., 37: 422-465. advantageous where breeders wish to keep two (or more) species as pure species. Due to cross incompatibility no new Smith, MM and McClly, ME (1978): A critical evaluation hybrids will be formed in nature and the species will remain of the specificity of aniline blue induced fluorescence. separate entities. Protoplasma, 95: 79-85

CONCLUSIONS Tyagi, AP, McComb" J and Considine, J (1991): Pollen germination percentage in three species showed that Cytogenetic and pollination studies in the Genus all the three species produced very high number of fertile Verticordia D.C., Aust. J. Bot., 39: 261-272. poUen and hence almost fully adapted to Fijian climatic conditions. Because all the three species are only partially Tyagi, AP, Considine, J and McComb, J (1992): self-compatible therefore it can safely be assumed that some Germination of Verticordia Pollen after storage at different sort of self-incompatibility mechanism is operating in these temperatures. Aust. J. Bot., 40: 151-155. species. None of the three species were cross compatible indicating that reproductive isolating mechanisms are in Tyagi, AP, Dass, CR, Nathan, S, Racule, T and Lakhan, S operation and therefore gene exchange is not possible (1995): Pollen fertility status in some flowering plant among these species. However to produce interspecific species of Fiji South Pac. J. Nat. Sci., 14: 211-222. hybrids, breeders have to look for other aids to facilitate fertilization and embryo rescue if embryo abortions occur Tyagi,AP and Singh, VV (1998): Pollen fertility and intraspecific and interspecific compatibility in mangroves of due to cross incompatibility. Fiji. Sex. Plant Reprod., 11 : 60-63.

REFERENCES Williams, W. (1951). Genetics of incompatibility in alsike clover, Trifolium hybridum. Heredity, 5: 51-71 Bid.well, RGS (1979): Plant Physiology. Collier MacMillan Ltd. Canada. pp 408-426.