Hybridization in Four Nigerian Physalis ( Linn . ) Species

Interspecific reciprocal crosses among four Nigerian Physalis species (Physalis angulata, P. micrantha, P. pubescens and P. peruviana) were carried out by transferring desired pollen grains to ovalate parents, bagged and labelled to prevent contamination by external pollen grains and for easy identification. Pollen cells of the F1 hybrid combination from the only viable cross (P. angulata x P. pubescens) were studied using standard cytogenetic techniques. The F1 hybrid obtained in the current investigation produced few flower buds and no matured fruit was harvested. Its average pollen grain diameter was found within the range of that of the two genitors and the pollen stainability was less than 50%. The hybrid was also characterized by meiotic irregularities. Based on the results obtained, the study concluded that P. pubescens is closely related to P. angulata and P. peruviana, while P. micrantha is more distant from them since the formal individuals were able to cross reciprocally among each other, though no matured fruit was produced except in one cross (P. angulata x P. pubescens).


Introduction
The genus Physalis Linn.species belongs to the night shade family called Solanaceae (Olorode, 1984;Jagatheeswari, 2014).This is a family of herbs, shrubs, rarely small trees, climbers, vines, epiphytes (Olorode, 1984).The genus includes about 100 species (Sultana et al., 2008;El-Sheikha et al., 2009) which were divided into three sections: Physalis (Euphysalis), Microphysalis and Megista (Sullivan, 1984).In West Africa, four species (P.angulata L., P. micrantha Link., P. pubescens L. and P. peruviana L.) were reported and all are present in Nigeria (Hutchinson and Dalziel, 1963;Olatunji, 1985).The genus constitutes a natural group of annual and perennial herbs characterised by solitary flowers borne on the axils of leaves and branches (Menzel, 1951;Olatunji, 1985;Shu, 1994).Olorode et al. (2013) opined that Physalis angulata L. (2n = 48) might be of hybrid origin from a cross between P. peruviana (2n = 24) and P. pubescens (2n = 24) based on their results from pollen grain, morphological and preliminary cytological studies.In addition, they observed that P. angulata has a wider latitudinal range of distribution with usually large populations, which is a characteristic of the distribution of successful hybrid genomes (Olorode et al., 2013).
There is no report on artificial hybridization among Nigerian Physalis so far, hence this study.The objective of the study was therefore to carry out hybridization among the four Nigerian Physalis species to elucidate the phylogenetic relationship that exists among them.

Germplasm collection and cultivation of Physalis species
Whole plants of all the Physalis species studied except P. micrantha were collected from different locations in South-Western Nigeria (Table 1 and Fig. 1).Accession numbers were given to the specimens, identified at the IFE Herbarium and voucher specimens were deposited.The whole plants collected were planted and nursed to maturity.Fruits of P. micrantha were also collected from different locations in South-Western Nigeria.The seeds were recovered from the fruits and planted separately.The seedlings were transplanted into 11-litre plastic buckets filled with top soil, in an experimental garden field beside the screen-house of the Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria.

Hybridization
Each of the four Physalis species was selfed before the hybridization was carried out, in order to determine their self-compatibility.Self-fertility test was carried out by randomly bagging twenty flower heads on plants of each species prior to the opening of the flowers.
Interspecific reciprocal crosses were carried out by physical emasculation of matured flower buds of each when the cell activities were considered to be at the peak (Jackson, 1962) and stored in 1:3 Acetic acid:alcohol.The anthers were later removed, squashed and stained in FLP Orcein by the squash technique according to Lasebikan and Olorode (1972).The pollen cells were examined and good meiotic chromosome spreads were photographed at x1000 under BK series system microscope.The pollen stainability was carried out according to the method of Olorode and Baquar (1976) using Cotton Blue in Lactophenol.
ovulate genitor before the occurrence of self-pollination.The pollen grains were transferred to the ovulate parent, bagged and labelled to prevent contamination by external pollen grains and for easy identification.
The crosses were monitored for fruit production and seed set.The F1 hybrid from the successful cross was raised along with its genitors and characterized for all morphological differences and similarities.

Meiotic chromosome study
Young flower buds from the F1 hybrid were collected for meiotic chromosome study between 9.00 am and 12.00 pm, 206

Results
All the four species studied were self-compatible.No natural hybrid was observed where P. angulata, P. pubescens and P. peruviana were growing sympatrically, whereas P. micrantha was observed to be growing allopatrically in ruderal and waste places.Of all the crosses that were carried out, including the reciprocal crosses (Table 2 and 3), no successful hybrid combination was obtained, except in one cross between P. angulata and P. pubescens (Fig. 2).Most of the fertilized ovaries were aborted few weeks after fertilization.Some fruiting calyces were enlarged with immature fruits which occasionally contained aborted seeds.A few full-sized seeds which failed to germinate were recovered in some fruits.
Some matured fruits with full-sized seeds were harvested.These seeds germinated; however, the F1 hybrid plants showed no influence of pollen donor.From P. angulata x P. pubescens (tetraploid x diploid), 15 seeds were recovered and all germinated.However, only one plant showed hybrid characteristics.Few flower buds were developed on this F1 hybrid plant and no matured fruit was harvested.
The pollen diameter of the hybrid plant (25.40-30.72 µm) fell within the range of that of the two genitors.The percentage pollen stainability of the F1 hybrid plant was 43.25% which was about 50% less than that of the parents.The flower colour, as well as the flower orientation, took after the staminate parent (Table 4).
The pollen cells of the F1 hybrid plant were characterized by meiotic irregularities (Fig. 3).The F1 hybrid plant was a triploid, having chromosome number of 2n = 36.The chromosomes were unequally distributed to the two poles at anaphase I; precocious chromosome movement and multiple laggards were observed at anaphase I and II.Unpaired chromosomes were also observed in some pollen mother cells.

Discussion
It was observed from the present study that the species that were found growing sympatrically produced no natural hybrids.The lack of hybrids among closely-related sympatrically-distributed species showed that strong preand post-zygotic isolated mechanism has developed among these species and therefore prevented hybridization (Pringle and Murry, 1991;Pascarella, 2007).Fourteen out of fifteen seeds recovered from P. angulata and P. pubescens that germinated did not show the influence of pollen donor.Menzel (1951) obtained no hybrid with P. angulata as a seed parent and also reported that no species combination 208 resulted in both matroclinal and hybrid populations they investigated, which was in contrast to what was observed in the F1 hybrid plants of P. angulata x P. pubescens in the present study.
In most crosses, heavy flower drop, early ovary abortion where fruiting calyces were slightly enlarged with or without ovary enlargement, as well as numerous aborted and few full-sized seeds, which failed to germinate, were observed.The unsuccessful hybridization recorded in the present study is similar to the report of Menzel (1951).Ganapathi et al. (1991) also recorded unsuccessful hybridization between P. pubescens and P. peruviana, and between P. pubescens and P. angulata including their reciprocal crosses.Such a result can be attributed to the fact that these species belong to different sections; even intrasectional crosses were sometimes difficult, as it was observed between P. pubescens and P. micrantha in the present study.The difficulties encountered over these crosses might also be due to the well-established barriers to hybridization in annuals (Menzel, 1951).
The embryo abortion observed in the study might be as a result of early nutrition barrier reported by Geerts et al. (2002) in the crosses between Phaseolus vulgaris and Phaseolus polyanthus.Moreover, the lack of coordination in endosperm may result in imbalanced production of growthrelating substances which in turn cause embryo abortion (Abbo and Ladizinsky, 1994).On the other hand, there might be incompatibilities between the parental genomes at cellular level which might have distorted the normal nuclear and cellular activities in the hybrid cell.Also, the parental genome may not function properly within the maternal cytoplasm (Pringle and Murry, 1991).
From the current study, one of the pollen mother cells of the hybrid obtained from the cross between P. angulata and P. pubescens showed 18 bivalents, which was a rare event.This association can be explained by the fact that P. pubescens designated "BB" was probably one of the genitors of P. angulata designated "AB" (Olorode et al., 2013).The hybrid might have undergone chromosome doubling resulting in AABB, restoring its fertility.Perhaps the restoration of fertility in P. angulata by polyploidization gave rise to regular segregation that was seen in P. angulata.By this same reasoning, the hybrid between P. angulata and P. pubescens was ABB.The two genitors shared similar genome, B known as pivotal genome (Kimber and Yen, 1988).Therefore, the homologous chromosomes in the B genome would have been able to pair, giving rise to 12 bivalents.The disruption in the meiotic mechanism governing bivalent pairing enabled the chromosomes of A genome to pair autosyndetically, producing 6 bivalents on a bascic number of x = 6.This is supported by the idea of Stebbins (1971) saying that the basic chromosome number of 12 and above in some genera and families might have evolved through chromosome doubling from groups with lower numbers.
The irregularities observed in the course of meiosis in the pollen cells of the F1 hybrid must have been responsible for malformation observed in some of the pollen grains, as well as the low pollen stainability recorded in the F1 hybrid plant.The F1 hybrid plant obtained from the present study failed to set fruit.This was similar to the observation of Oziegbe and Faluyi (2011) in a hybrid obtained from tetraploid and diploid crosses in Ludwigia.This observation was attributed to genetically imbalanced gametes that originated from the parents of the F1 hybrid which eventually led to hybrid sterility observed (Oziegbe and Faluyi, 2011).

Conclusions
It can be concluded from the persent study that P. pubescens is closely related to P. angulata and P. peruviana, while P. micrantha is more distant from them.This is because P. pubescens was able to cross reciprocally with both P. angulata and P. peruviana even though fruits were not set, except in the cross between P. angulata and P. pubescens.

Fig. 2 .
Fig. 2. F 1 Hybrid between P. angulata x P. pubescens A. The habit of the F 1 hybrid plant (Scale = 8 cm); B. Fertilized ovary which slightly enlarged and later dropped after few days (arrowed) (Scale = 3 cm); C. Flower of the hybrid plant (Scale = 0.25 cm); D. Leaf of P. angulata; E. Leaf of F 1 hybrid plant; F. Leaf of P. pubescens (all Scale = 2 cm)

Table 2 .
The crosses involving the four Physalis species studied, including the reciprocal crosses

Table 3 .
The summary of the crosses in relation to the percentage of fertilized ovules and fruit set

Table 4 .
Characteristics of the F1 hybrid plants and its genitors *The end of life cycle was taken when the leaves started drying up