Pre-fertilization Barrier, Crossability and Meiotic Behavior of Interspecific Hybrids Among Brassica Species


 Inter-specific hybridization is an important driving force in plant evolution, speciation and creation of novel genetic variations those are not available with the naturally occurring species. Present investigation includes study of pre-fertilization barriers, phenotypic characterization and the meiotic abnormalities during microsporogenesis in the interspecific hybrids. Mean pollen tube germination percent in interspecific hybrids was recorded to be highest in case of B. carinata x B. nigra (33.67±11.35) and lowest between B. carinata x B. napus (18.68±6.62) whereas in the reciprocal crosses it was highest for B. napus x B. carinata (33.06±13.41). Siliqua set ranged from 33% in B. carinata x B. napus to 72% in selfedB. carinata. Seed set % ranged from 0% in B. rapa x B. carinata and B.nigra x B.carinatato 25.64 % for B. carinata x B.nigra. Mean pollen tube growth was recorded highest in B. carinata x B. nigra (21.23±5.33) while was lowest in B. carinata x B. napus (10.18±3.17) and among reciprocals it was highest for B. nigra x B. carinata (21.39±7.53). Pollen tube abnormalities viz., coiling and bending of pollen tube, hairpin shaped pollen tube, swelling of tube tip, tube bifurcation, more than one tube emerging from pollen, tubes growing in wrong direction were observed. B. carinata as pollen parent showed highest mean pollen tube abnormality percent with B. napus (36.32±8.62) whereas lowest with B. nigra (18.09±6.73). It was observed highest with B. rapa (49.170 ±13.55) when B. carinata was used as pollen parent. Some of the pre fertilization barriers related to stylar and ovule region can also be observed through fluroscent microscopy. As per the observations B. carinata performed better as a seed parent with B. nigra, B. juncea and B. rapa whereas, with B. napus it performed good as a pollen parent. Correlation study gave the association the above traits with each other. The meiotic abnormalities showed improper alignment at M-I, M-II, A-I, A-II and Telophase.


Introduction
Brassica is one of the genuses belonging to the family Brassicaceae and includes crops of agricultural and horticultural importance. Out the species under genus Brassica, six are of economic importance viz., B.rapa (AA), B.nigra (BB), B.oleraceae (CC), B.juncea (AABB), B.napus (AACC), and B.carinata (BBCC). Out of these, the diploid species of Brassica are considered to be the progenitor species or primary species whose natural cross combinations followed by chromosome doubling resulted in evolution of three amphidiploids secondary species. The genetic relationship between these primary and secondary species was depicted through U's Triangle Model (Nagaharu U., 1935). Rapeseed mustard is one of the most important oilseed crop grown worldwide. Oilseed Brassica is the second most important edible oilseed crop in India after soybean and accounts for nearly one third of the oil produced in India, making it the country's key edible oilseed crop. Wide or distant hybridization is a mating between individuals of different species or genera. It combines different genomes into one nucleus and thus results in changes in genotypes and phenotypes of the progenies. Since, the rst ever But not all of the crosses are successful, the cross incompatibility in the interspeci c hybridizations has impeded the development of hybrids. Many efforts have been made to improve the Brassica crops by sexual crosses intended to enrich the germplasm through arti cial resynthesis and wide hybridization of naturally existing amphidiploids. With due course of time, the cultivated primary species have been improved to a great extent for their agronomic performance and their genome have also been modi ed considerably. Similarly the primary genome residing in the amphidilpoids have also been undergone cytological changes and the modi ed. Therefore interspeci c hybridization among the existing primary and secondary species might bring some novel variant that enrich the available germplasm. However, even though there is large tolerance for interspeci c hybridization between the Brassica crop species and several of the wild species related to the Brassica species, successful use of sexual hybridization is limited. A number of preand post-fertilization barriers limit the exploration of diversity of different genomic combination. Depending upon the types of barriers several methods have been used to overcome these barriers.

Material And Methods
Five species of Brassica were used for interspeci c hybridization i.e., B. rapa, B. nigra, B. juncea, B. napus, B. carinata. Eight buds per raceme were emasculated in the evening hours and were then pollinated the next day in the morning hours. All the possible crosses among the 5 species were made taking the reciprocals also into consideration. Sel ng of all the 5 species was also carried out. For understanding pollen pistil interaction pre-fertilization studies were made for which bright eld microscopy and uorescent microscopy at different intervals after pollination were carried out. For bright eld microscopy the pistil were removed from the raceme carefully and were kept in 1N HCL for 10 min. After that the buds were rinsed with distilled water and stained with 1% aniline blue for 5-15sec. After staining the samples were put in destaining solution for 30 min. After destaining the samples were rinsed with distilled water and mounted on pure lactic acid. For uroscent microscopy Aniline Blue was used as a callose staining urochrome (Dumas and Knox, 1983). The xed samples were rinsed with distilled water and after careful maceration squash preparation was done. Prior to squash preparation the samples were kept in 8N NaOH solution for about 2 to 6 hours for softening. The samples were then stained in Decolourised Aniline Blue (DAB) solution for 30min to 1 hour. Parameters viz., Pollen germination percent, mean pollen tube growth and pollen tube abnormality percent were assessed. Siliqua set percent and seed set percent were also recorded for assessing cross compatibility relationship. Morphological studies of the hybrids were also done in the eld along with the parents and observation on different morphological characters was taken. Meiosis study of the F1s was also carried out and for this the buds were collected in the morning and were xed in Carnoy's solution for 24 hours. After that the samples were transferred to 70% ethanol and refrigerated till further use. For slide preparation the anther was crushed and one percent acetocarmine was used to stain it. Correlation between the crossability parameters was also studied.

(a) Cross compatibility relationship:
Crossability relationship among brassica species was determined on the basis of successful siliqua and seed set percent. The direct inter-speci c cross involving diploid and amphidiploids and their reciprocals were included for the study. Siliqua set percent in inter-speci c hybrids ranged from 21-61%. It was recorded highest in the interspeci c cross of B. napus × B. juncea (61%) whereas lowest in the interspeci c cross of B. nigra × B. napus (21%). In inter-speci c crosses seed set percent ranged from 0.00 % to 28.89%. Highest seed set percent was recorded in the cross B. juncea × B. napus (28.89%). However, no seed set was observed in the crosses of B. rapa × B. napus, B. rapa ×B. carinata, B. nigra × B. carinata, B. nigra × B. napus ( Table 1). The crossability relationship based on seed set percentage is depicted in Fig. 1. It is re ected from the results that in case diploid and amphidiploids interspeci c crosses mostly the higher siliqua and seed set were observed where the amphidiploids were taken as female parent. In most of the interspeci c crosses involving amphidiploids higher seed and siliqua set was observed when a parent with higher chromosome number is taken as female. In the crosses where only diploids were used i.e. B. rapa and B. nigra similar effect of female parent was found. Higher siliqua and seed set has been observed in cross having B. nigra as female parent. Signi cant role of female parent in controlling the seed set has also been reported by saba (2012). The genome a nity during interspeci c hybridization is key factor for the success of crosses. The genomes that are close and common gave better    Bright eld microscopy Bright eld microscopy and uorescent microscopy was carried out to study the pre-fertilization in different inter-speci c crosses. The buds xed under different time intervals of pollination were subjected to microscopy. Through Bright eld microscopy, three parameters were estimated viz., pollen germination percent, pollen tube growth and pollen tube abnormality percent. In the self-pollinated samples pollen grains started showing germination 30 minutes after pollination and highest pollen germination percentage at 30 minutes was recorded for B. nigra self (8.0%), while in inter-speci c hybrids the pollen germination started 2 hrs or 8 hrs after pollination. The rate of pollen tube growth was almost constant when measured at 2 hrs of pollination and Fluorescent Microscopy: For studying the pollen tube growth in stylar region uorescent microscopy was done at different time intervals for each and every cross. Breaking of pollen tube in upper half of style, twisting of pollen tubes in lower half of style and pollen tubes not entering the ovules but passing through the gaps in between the ovules were some of the pre fertilization barriers recorded (Fig. 3).

(c) Correlation Studies:
Correlation studies were carried out between different parameters of crossability and prefertilization barriers, so to determine the relationship between them. The pollen germination percent showed highly signi cant and positive correlation with mean pollen tube growth, and percent siliqua set and highly signi cant and negative correlation with pollen tube abnormality percent.A highly signi cant and positive correlation also existed between mean pollen tube growth and siliqua set percent and a highly signi cant and negative correlation existed between mean pollen tube growth and pollen tube abnormality percent.Pollen tube abnormality percent had a highly signi cant and negative correlation with all of the crossability parameters with lack of correspondence with seed set percent.Siliqua set percent was found in correspondence with percent seed set. A nity of A genome with C genome is more pronounced than a nity of A genome with B or B genome with C genome. It was reported in the literature that during evolution A and C genome had evolved from the same lineage while the B genome has taken a different route. That is why the B genome has showed low a nity with both the two genome (Kaminski et al., 2020). both of the parents. The siliqua size of hybrids was smaller than that of parents, with poor seed set. The hybrids were partially sterile and so no seed set upon sel ng. Seeds were recovered only from open pollination (Fig. 5a). B.carinata x B. rapa F 1 s were similar to the female parent in branching pattern and growth habit, were tall having thick and waxy stem. Siliqua set was more than parents whereas seed set was lower (Fig. 5b). The cross of B. carinata x B. napus was like female parent in terms of growth habbit. Waxy stem and tall height was observed (Fig. 5c). Growth habit was like that of male parent, profused and appressed siliqua were observed and reduced siliqua size as compared to the female parent in B.carinata x B.nigra cross (Fig. 5d). B.napus x B.carinata F 1 s were closely resembled the female parent in plant type and growth habit. Pod and pod bearing habit was also like that of female parent (Fig. 5e). In B. napus × B. juncea cross the hybrids resembled the female parent in plant type and growth habit. The pods of the hybrids and its pod bearing habit also resembled the female parent B. napus (Fig. 5f). The

Discussion
The percent pollen germination in self was early as compared to the interspeci c hybrids (Sridevi and Sarla, 1996). Pollen tube growth was fast in crosses where there was genome sharing between crosses. Rate of pollen tube growth was almost constant after 2 hrs and 8hrs of pollination. An increase in the growth of pollen tube was observed from 24 hrs to 48hrs of pollination. Pollen tube abnormality percent was high in distant crosses.The type of abnormality was not speci c to a particular cross but all of the above mentioned abnormalities were recorded in each of the interspeci c crosses