Journal of Heredity Advance Access published online on March 13, 2008
Journal of Heredity, doi:10.1093/jhered/esn019
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Brief Communications |
Inheritance of Pollen-less Anthers and "Thrum" and "Pin" Flowers in Periwinkle
From the Central Institute of Medicinal and Aromatic Plants, Resource Centre, Allalasandra, Bangalore 560 065, India
Address correspondence to R. N. Kulkarni at the address above, or e-mail: krnpbg{at}yahoo.co.in.
Two mutants, 1 with small, pollen-less anthers (OR-EA) and another with "pin" flowers (EMS 13-2), in contrast to "thrum" flowers found in normal periwinkle (Catharanthus roseus) plants, were isolated after induced mutagenesis in strain OR and cultivar, "Dhawal," respectively. Inheritance of these 2 traits, pollen-less anthers, and pin flowers was studied by crossing the mutants with their respective parental strains. Segregation ratios observed in F2 and testcross generations of the cross OR-EA x OR suggested that the pollen-less anthers trait was determined by duplicate recessive genes. Data obtained from F2 and F3 generations of the cross involving mutant EMS 13-2 with pin flowers and its parental variety Dhawal, suggested that production of pin (mutant) and thrum (normal) flowers was under the control of inhibitory epistatic interaction between 2 independently inherited genes.
Madagascar periwinkle (Catharanthus roseus [L.] G. Don) is commonly grown as a garden plant for its variously colored flowers and heat and drought tolerance. It is also highly valued for its anticancer (vincristine and vinblastine) and antihypertension (ajmalicine) alkaloids present in its leaves and roots, respectively. Because of the high value but relatively low concentrations of these alkaloids, periwinkle has become one of the most intensively investigated medicinal plants after the discovery of these alkaloids in 1950s. On average, about 70 research papers were published each year on various aspects of this plant during the period 1950–2001, indicating continued interest in this plant (van der Heijden et al. 2004). However, as is evident from recent reviews (Sreevalli 2002; van der Heijden et al. 2004), relatively very little work has been done on the genetics and breeding aspects of this plant.
Until recently, periwinkle was considered as an autogamous species (see Kulkarni et al. 2005), although Darwin and Delpino long ago reported that selfing within individual periwinkle flowers is not automatic and that pollination typically occurs through nectar-seeking insects (Knuth 1909; Rendle 1971). Recent studies have confirmed these observations, further revealing that geitonogamy and phenotypic assortative mating for flower color brought about by pollinating butterflies give a false impression that periwinkle is autogamous, when flower color is used as a marker trait to determine the breeding system (Kulkarni 1999; Sreevalli et al. 2000). Kulkarni (1999) reported an outcrossing rate of 79% when marker traits other than flower color were evaluated.
In allogamous species, hybrid varieties are often developed if significant heterosis is found for economically important traits. Significant heterosis for leaf and root yield has been reported in both inter- and intraspecific hybrids in periwinkle (Levy et al. 1983; Sevestre-Rigouzzo et al. 1993). Although periwinkle is a cross-pollinating species, the development of hybrid varieties has been hampered by the lack of a male-sterility system that can be manipulated by plant breeders. Levy et al. (1983) therefore suggested the development of pure line cultivars until male-sterile lines become available. Sevestre-Rigouzzo et al. (1993) suggested the use of micropropagation to exploit the hybrid vigor of heterotic hybrids. Utilization of high levels of outcrossing and the absence of intraflower self-pollination combined with the use of seedling markers for identifying hybrids has also been suggested for exploiting heterosis (Kulkarni 1999). Sreevalli et al. (2003) identified a functional "male-sterile" mutant in which anthers failed to dehisce and demonstrated its utility in the production of hybrid seeds. The mutant could be multiplied through artificial selfing or vegetatively through stem cuttings or micropropagation; its utility, however, would be determined by economics of its multiplication, production of hybrid seeds, and heterotic advantage of the resulting hybrids.
Recently, Kulkarni et al. (2001, 2005) reported the existence of a few uncommon autogamous strains in periwinkle and described the mechanism and genetics of their self-pollination. In normal allogamous strains, the stigma remained below the base of the anthers until flower drop, whereas in autogamous strains, self-pollination occurred due to an increase in the length of the gynecium brought about by either an increase in the length of the style or of the ovary. Self-pollination was found to be under the control of duplicate recessive genes. Although this variation in floral structure with respect to gynecium length resembled heteromorphy, there was no heteromorphy for anther position. According to Al Wadi and Richards (1993), anther position monomorphy and self-compatibility may represent an intermediate condition in the evolution of full distyly.
After induced mutagenesis, we identified a pollen-less male-sterile mutant and a long-styled mutant exhibiting stigma position above the anthers ("pin" flowers) in contrast to the stigma position below the anthers ("thrum" flowers) found in the normal allogamous strains, suggesting complete distyly with reciprocal anther and stigma positions. These 2 mutants appeared to be potentially useful for utilizing heterosis and understanding the pollination system in periwinkle. The mode of inheritance of these 2 mutant traits (pollen-less anthers and pin flowers) is reported here.
 |
Materials and Methods |
|---|
 Top Materials and Methods Results and DiscussionReferences |
|---|
Two mutants, 1 with pollen-less anthers (OR-EA) and another with thick abnormal leaves and pin flowers, that is, with stigmas above the anthers (EMS 13-2), were isolated from M2 generations raised from ethyl methanesulphonate (EMS) treated seeds of a strain OR and the cultivar, "Dhawal," respectively. Seeds presoaked in water for 19 h had been treated with unbuffered 0.6% aqueous solution of EMS for 4 h. Parental strain OR is an autogamous strain in which self-pollination occurs due to increase in length of the style, whereas Dhawal is a normal allogamous cultivar with thrum flowers, that is, stigmas below the anthers. Dhawal is an induced mutant with higher content of total alkaloids in the leaves developed from cultivar "Nirmal."
The mutants along with plants of their parental lines were raised in pots in the glasshouse and studied for their anther characteristics and stigma position. Anthers, from flower buds fixed in ethyl alcohol–glacial acetic acid mixture (3:1) 1 day before anthesis, were hydrolyzed in 0.8 N NaOH for 5 min. Each anther was then mounted on a microscopic slide in 1–2 drops of lactophenol and covered gently with a cover slip without rupturing the anther. Anther length and width (at the middle region) were measured under the microscope using a micrometer. Similarly, hydrolyzed anthers were smeared in a drop of 1% acetocarmine and the total number of pollen grains present each anther was counted by scanning the entire slide under the microscope. Pollen stainability with 1% acetocarmine was also determined separately. Pollen germination was determined using 0.1% water–agar containing 2% sucrose. Stigma position relative to anther base in OR, OR-EA, and Dhawal was determined as the difference between gynecium length and height of anther base in the corolla tube (Kulkarni et al. 2005). The position of stigma relative to tip of anthers in EMS 13-2 was determined by measuring the height of the sigma above the tip of anthers.
To determine the mode of inheritance of mutant traits, mutants were crossed to respective parental lines and plants of F1, F2, and F3 or testcross generations were raised from seeds produced via artificial hybridization or selfing. The segregating generations were scored for plants with normal or mutant traits. Chi-square tests were used to test the goodness-of-fit of observed frequencies of different phenotypic classes in the F2, F3, and testcross generations to expectations based on simple Mendelian models.
The F2 and testcross generations of the cross OR-EA x OR were raised in the glasshouse and scored for male-fertile and male-sterile plants. Because OR was a self-pollinating strain, male-sterile plants in the F2 and testcross generations could be easily identified as they failed to produce fruits in the glasshouse. All plants including male-sterile plants were then checked for their anther size to identify plants with normal-sized anthers without pollen grains and those with small anthers with pollen grains.
The F2 generation of the cross EMS 13-2 x Dhawal consisting of 305 plants was raised in the field, and the plants were scored for normal and mutant traits of EMS 13-2. Flowers of all plants were examined for the position of the stigma in their corolla tubes. Corolla lobes of 5 tagged flowers of each of the F2 plants were removed (to prevent pollination by butterflies) to determine fruit set resulting from autogamy due to stigma height intermediate to parental stigma heights, if any. Twenty-five randomly selected normal F2 plants were transferred to the glasshouse and artificially selfed to produce F3 seeds, and their segregation behavior was studied in the F3 generation.
|
Results and Discussion |
|---|
|
TopMaterials and MethodsResults and DiscussionReferences |
|---|
The mutant OR-EA had smaller anthers than did OR, and 96% of its anthers lacked pollen grains. Only 4% of anthers showed a few pollen grains; on an average 3 pollen grains per anther in contrast to 1134 pollen grains per anther OR (Table 1). F1 progeny of the cross OR-EA x OR showed normal-sized anthers that were fully fertile as suggested by their high pollen stainability with 1% acetocarmine and normal fruit set like parent OR. The F2 and testcross (OR-EA x F1) generations of the cross OR-EA x OR segregated into male-fertile and male-sterile (with small, pollen-less anthers) plants in the ratio of 15:1 and 3:1, respectively (Table 2), suggesting that male sterility expressed as pollen-less anthers was conditioned by duplicate recessive genes. However, neither plants with normal-sized anthers without pollen grains nor those with small anthers containing pollen grains were observed in the studied populations consisting of 89 and 26 plants in the F2 and testcross generations, respectively, suggesting that small anther size and lack of production of pollen grains were probably due to pleiotropic effects of the same genes. Gene symbols ms1 and ms2 are proposed for duplicate recessive genes governing the type of male sterility found in the mutant periwinkle line OR-EA. Similar mutants with pollen-less anthers have been reported in soybean (Palmer 2000), pigeon pea (Saxena and Kumar 2001), and African night shade (Ojiewo et al. 2005). This type of male sterility was found to be governed by a single recessive gene in pigeon pea (Saxena and Kumar 2001) and soybean (Palmer 2000). In pigeon pea, it was also characterized by reduced anther size, as found in our periwinkle mutant OR-EA. In Asiatic hybrid, lily pollen-less anthers’ trait has been found to be determined by the interaction between cytoplasmic and nuclear genes (Yamagishi 2003). Mutants with pollen-less anthers resulting from apparent meiotic defects and/or abnormalities in cell layers surrounding locules with pollen sacs completely missing in advanced stage of anther development have been reported in Arabidopsis thaliana (Sanders et al. 1999). In another mutant, a premeiotic mutant sporocyteless/nozzle (spl/nzz) of A. thaliana, differentiation of primary sporogenous cells into microsporocytes and anther wall formation is blocked, and at anthesis, anthers are composed of highly vacuolated parenchyma cells (Yang et al. 1999). Recently, Wijeratne et al. (2007) studied differential expression of genes in anthers of wild-type plants and 2 A. thaliana mutants spl/nzz and ems1 (excess microsporcytes1) and found that a total of 1954 genes were differentially expressed in the 2 mutant anthers.
|
|
The mutant EMS 13-2 had thick abnormal leaves as compared with parental variety, Dhawal, and a majority of its flowers had 6 corolla lobes instead of 5 as found in normal periwinkle plants (Table 1). The position of the stigma in the corolla tube was above the cone of anthers, that is, the mutant produced pin flowers (Figure 1), in contrast to thrum flowers found in normal periwinkle plants and in parental variety Dhawal, where stigma was below the base of anthers (Figure 2). Anthers were indehiscent, and pollen stainablity with acetocarmine (1%) stain was high, but pollen germination was very low (Table 1). Many staining tests using nonvital stains such as acetocarmine, aniline blue in lactophenol, iodine, acid fuchsin, and Alexander’s stain (Alexander 1969) are satisfactory in assessing pollen sterility but are not dependable for testing viability as they stain both viable and nonviable pollen (Janssen and Hermsen 1976; Shivanna 2003; Nyine and Pillay 2007). Several artificial self-pollinations and reciprocal crosses were made with the parental variety. Only 1 fruit with 2 seeds was obtained after more than 300 crosses were made using the mutant as female parent and parental variety Dhawal as male parent. Only 1 F1 plant could be obtained from 2 seeds obtained from this fruit as the other seed failed to germinate. Thus, low pollen viability as indicated by in vitro pollen germination and poor fruit and seed set after artificial self- and cross-pollination with parental cultivar Dhawal suggested high male as well as female sterility in the mutant EMS 13-2. The F1 plant showed normal phenotype and resembled the parental variety, Dhawal. It was fully fertile as it showed normal fruit set on artificial self-pollination. A F2 population consisting of 305 plants was raised in the field and scored. The observed ratio of plants with normal and mutant phenotypes in the F2 generation conformed to a 13:3 ratio (Table 3). Twenty-five randomly selected normal F2 plants were transferred to the glasshouse and artificially selfed to produce F3 seeds. Due to mortality of some F2 parents, progenies of only 13 F2 plants could be raised in the field and scored for normal and mutant plants. Five out of these 13 F2 progenies were nonsegregating and produced only normal plants, whereas 8 segregated for normal and mutant plants. The segregation ratio in 5 of these 8 segregating F2 progenies conformed to a 13:3 ratio (Table 3, plants 6–9 and 11), whereas 2 of these progenies conformed to a 3:1 ratio (Table 3, plants 12 and 13). The segregation ratio for plant 10 (Table 3) conformed to both 3:1 and 13:3 ratios but more closely fitted the 13:3 ratio. Based on the model of genetic control detailed below, in the F3 generation, 7/13 of normal F2 plants are expected to show no segregation, whereas 4/13 and 2/13 plants are expected to segregate in 13:3 and 3:1, ratios, respectively. Thus, segregation data of F2 progenies in F3 generation appeared to confirm the 13:3 ratio of normal:mutant plants observed in the F2 generation. None of the F2 plants showed recombination between any of the mutant traits, that is, leaf shape, stigma height with respect to anthers, number of corolla lobes, and male and female sterility. The mutant traits appeared to be either very tightly linked or governed by a pleiotropic dominant allele whose expression was inhibited by a dominant allele of another independently inherited gene. Thus, distyly observed in flowers of normal parental (thrum) and mutant (pin) plants appeared to be determined by an inhibitory, epistatic interaction between 2 independent genes. Gene symbols P and T are proposed for genes involved in the production of mutant pin and normal thrum type of flowers, respectively, in periwinkle, with gene T being inhibitory to gene P. Accordingly, genotypes of plants producing normal thrum flowers would be P-T-, T-pp, or pptt, whereas of those producing mutant pin flowers would be P-tt. There appears to be no report of heterostyly in periwinkle. However, a short-styled mutant conditioned by a single recessive gene hsf with pleiotropic effects on a number of other vegetative and reproductive characters has been reported in periwinkle (Mishra and Kumar 2003a). They have also reported another mutant with bigger flowers and varying number of flower organs conditioned by a single recessive pleiotropic gene fos, which also affected several other vegetative and reproductive traits (Mishra and Kumar 2003b).
|
|
|
In Amsinckia, distyly is controlled by a single locus with 2 alleles, S and s, and all species are self-compatible (Ganders 1975). Tristyly (a heteromorphic heterostyly condition in which short-styled, mid-styled, and long-styled morphs with reciprocal anther and stigma positions coexist) in Lythrum junceum Bank and Sol. is controlled by the epistatic interaction of 2 independent genes S and M, each with 2 alleles, with genotypes mmss and M-ss determining long- and midstyled morphs, respectively, whereas genotypes MMSS, MMSs, MmSs, and mmS- determining the short-styled morph (Frankel and Galun 1977). The mutant traits in the present mutant of periwinkle (EMS 13-2) viz., abnormal leaves, abnormal corolla (6 instead of 5 corolla lobes), long style, indehiscent anthers, and high male and female sterility, appeared to be similar to those described by Gottschalk (1987) in the dim-segment of the Pisum genome. Mutations in 5 closely linked genes in the dim-segment ac, gfc, stpr, dim, and ster determining abnormal corolla, green flower color, abnormal sex organs, narrow leaves, and female sterility appeared to be inherited together. Similarly, in Primula L., an extensively studied heterostyly system, the heterostyly super gene S, consists of 6 closely linked loci viz., G, S, Is, Ip, P, and A that control style length, stigmatic surface, stylar incompatibility, pollen incompatibility, pollen size, and anther height, respectively. It is responsible for only 0.005 of the length of the S chromosome (de Nettancourt 2001).
The purpose of studying this cross was to isolate recombinants combining pollen fertility with stigma height intermediate between those of normal and mutant plants, so that the anthers dehisce on the stigma within the corolla tube and thus result in automatic self-pollination. However, no such recombinants, in which automatic self-pollination occurred, were found. If traits found in the mutant, such as stigma position above the anthers and high male and female sterility are not due to pleiotropy, then it might be possible to generate rare recombinants if a much larger F2 population can be evaluated. Given the challenges in generating progeny from our mutant line EMS 13-2, this may be quite difficult.
|
Acknowledgments |
|---|
The authors thank Dr. S. P. S. Khanuja, the Director of the Central Institute of Medicinal and Aromatic Plants, Lucknow, India, for facilities and encouragement. The authors also thank 3 anonymous reviewers for their critical review of the earlier version of this paper and useful suggestions.
|
Footnotes |
|---|
Corresponding Editor: Reid Palmer
Received September 17, 2007
Accepted
|
References |
|---|
|
TopMaterials and MethodsResults and DiscussionReferences |
|---|
-
Alexander MP. Differential staining of aborted and non-aborted pollen. Stain Technol. (1969) 44:117–122.[Web of Science][Medline]
Al Wadi H, Richards AJ. Primary homostyly in Primula L. subgenus Sphondylia (Duby) Rupr. and the evolution of distyly in Primula. New Phytol. (1993) 124:329–338.[CrossRef][Web of Science]
de Nettancourt D. Incompatibility and incongruity in wild and cultivated plants (2001) Berlin (Germany): Springer.
Frankel R, Galun E. Pollination mechanisms, reproduction and plant breeding (1977) Berlin (Germany): Springer.
Ganders FR. Mating patterns in self-incompatible distylous populations of Amsinckia (Boraginaceae). Can J Bot. (1975) 53:773–779.
Gottschalk W. The genetic basis of variation. In: Improving vegetatively propagated crops—Abbott AJ, Atkin RK, eds. (1987) London: Academic Press. 317–334.
Janssen AWB, Hermsen JGTh. Estimating pollen fertility in Solanum species and haploids. Euphytica. (1976) 25:577–586.[Medline]
Knuth R. Handbook of flower pollination. (1909) III. Oxford (UK): Clarendon Press.
Kulkarni RN. Evidence for phenotypic assortative mating for flower colour in periwinkle. Plant Breed. (1999) 118:561–564.[CrossRef]
Kulkarni RN, Sreevalli Y, Baskaran K. Allelic differences at two loci govern different mechanisms of intra-flower self-pollination in self-pollinating strains of periwinkle. J Hered. (2005) 96:71–77.
Kulkarni RN, Sreevalli Y, Baskaran K, Kumar S. The mechanism and inheritance of intra-flower self-pollination in self-pollinating variant strains of periwinkle. Plant Breed. (2001) 120:247–250.[CrossRef]
Levy A, Milo J, Ashri A, Palevitch D. Heterosis and correlation analysis of vegetative components and ajmalicine contents in the roots of the medicinal plant Catharanthus roseus (L) G. Don. Euphytica. (1983) 32:557–564.[CrossRef][Web of Science]
Mishra P, Kumar S. Manifestation of heterostyle character by induction of recessive hsf mutation responsible for thrum type herkogamous flowers in Catharanthus roseus. J Med Arom Plant Sci. (2003a) 25:2–7.
Mishra P, Kumar S. Flower organ size (FOS) gene in Catharanthus roseus with pleiotropic roles in flower growth. J Med Arom Plant Sci. (2003b) 25:705–711.
Nyine M, Pillay M. Banana nectar as a medium for testing pollen viability and germination in Musa. Afr J Biotechnol. (2007) 6:1175–1180.
Ojiewo CO, Agong SG, Murakami K, Tanaka A, Hase Y, Masuda M. Male-sterility induced by gamma-ray irradiation of African nightshade (Solanum nigrum L. spp. villosum) seed. J Hortic Sci Biotechnol. (2005) 80:699–704.
Palmer RG. Genetics of four male-sterile, female-fertile soybean mutants. Crop Sci. (2000) 40:78–83.
Rendle AB. The classification of flowering plants II (1971) Cambridge (UK): Cambridge University Press.
Sanders PM, Bui AQ, Waterings K, McIntire KN, Hsu Y, Lee PY, Truong MT, Beals TP, Goldberg RB. Anther development defects in Arabidopsis thaliana male sterile mutants. Sex Plant Reprod. (1999) 11:297–322.[CrossRef]
Saxena KB, Kumar RV. Genetics of a new male-sterility locus in pigeonpea (Cajanus cajan [L.] Millsp). J Hered. (2001) 92:437–439.
Sevestre-Rigouzzo M, Nef-Campa C, Ghesquiere A, Chrestin H. Genetic diversity and alkaloid production in Catharanthus roseus, C. trichophyllus and their hybrids. Euphytica. (1993) 32:151–159.[CrossRef]
Shivanna KR. Pollen biology and biotechnology (2003) Enfield (NH): Science Publishers Inc.
Sreevalli Y. Inheritance of some morphological, floral, reproductive and economically important traits in the medicinal plant, periwinkle [Catharanthus roseus (L) G. Don] [PhD dissertation] (2002) [Bangalore (India)]: Bangalore University.
Sreevalli Y, Baskaran K, Kulkarni RN. Inheritance of functional male sterility in the medicinal plant, periwinkle. Indian J Genet. (2003) 63:365–366.
Sreevalli Y, Baskaran K, Kulkarni RN, Kumar S. Further evidence for the absence of automatic and intra-flower self-pollination in periwinkle. Curr Sci. (2000) 79:1648–1649.[Web of Science]
van der Heijden R, Jacobs DI, Snoeijer W, Hallard D, Verpoorte R. The Catharanthus alkaloids: pharmacognosy and biotechnology. Curr Med Chem. (2004) 11:1241–1253.[Medline]
Wijeratne AJ, Zhang W, Sun Y, Liu W, Albert R, Zheng Z, Oppenheimer DG, Zhao D, Ma H. Differential gene expression in Arabidopsis wild-type and mutant anthers: insights into anther cell differentiation and regulatory networks. Plant J. (2007) 52:14–29.[CrossRef][Web of Science][Medline]
Yamagishi M. A genetic model for a pollenless trait in Asiatic hybrid lily and its utilization for breeding. Sci Hortic. (2003) 98:293–297.[CrossRef]
Yang WC, Ye D, Xu J, Sundaresan V. The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein. Genes Dev. (1999) 13:2108–2117.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||