© 2015 The Japan Mendel Society Cytologia 80(1): 37–43 Development and Characterization of a Hexaploid Pennisetum orientale (2n=6x=54) Cytotype Recovered through BIII Hybridization Pankaj Kaushal*, Krishna K. Dwivedi, Auji Radhakrishna, Manoj K. Srivastava, Devendra R. Malaviya, Ajoy K. Roy, Saurabh Saxena and Sharmishtha Paul Crop Improvement Division, Indian Grassland and Fodder Research Institute, Jhansi 284003, India Received July 7, 2014; accepted October 8, 2014 Summary Pennisetum orientale is an important perennial species having high ecological and forage value. It is largely tetraploid (2n=4x=36) and obligate apomictic in mode of reproduction. A new hexaploid (2n=6x=54) derivate of P. orientale is reported here, recovered in a population derived from self-pollination of a tetraploid genotype. The origin of the 6x cytotype from a 4x cytotype is demonstrated through fertilization of an unreduced egg cell with a reduced male gamete (BIII hybridization), yielding experimental evidence of uncoupling of apomixis components in P. orientale. This 6x cytotype was compared for its morphological, cytological and reproductive traits vis-à-vis parental 4x cytotype, and also for potential of gene transfer in otherwise apomictic P. orientale. Key words Apomixis, Cytotype, Hexaploid, Pennisetum, P. orientale. Pennisetum L. Rich is one of the largest genera (tribe Paniceae) of Gramineae (Poaceae) family and encompasses several economically important crops species. It is a polybasic genus consisting of over 140 species and represented by x=5, 7, 8 or 9 basic chromosome number and 2x to 8x ploidy levels (Jauhar 1981). Cultivated pearl millet (P. glaucum) is the most important species of this genus being utilized as food and fodder crop. P. pedicellatum, P. purpureum, P. orientale and pearl millet-napier grass (P. purpureum) hybrids are the other popular fodder crops in semi- arid tropics. Other wild species belonging to secondary and tertiary gene pools of pearl millet are also important as donors of desirable traits for pearl millet improvement (Hanna 1986). P. orientale belongs to the tertiary gene pool and is a valuable pasture grass with high palatability, nutritive value and drought tolerance. It is a tall rhizomatous, deep rooted and perennial grass known for its excellent soil binding qualities (Whyte et al. 1959, Bor 1960). P. orientale was also utilized for introgressing perenniality, stress tolerance and apomixis in cultivated pearl millet following interspecific hybridization approach. Most of the hybrids were sterile owing to major genomic differences between the two species. However, the hybrids generated appreciable information on genome relatedness between the two species (Dujardin and Hanna 1983, Hanna and Dujardin 1982, Zadoo and Singh 1986, Patil and Singh 1964, Kaushal et al. 2010) as well as molecular studies on evolution of genomes and mode of reproduction (Akiyama et al. 2011, Sahu et al. 2012). P. orientale has a basic chromosome number x=9 with tetraploid cytotypes (2n=4x=36) as most predominant forms. Other ploidy levels, such as 2x, 3x, 5x, and 6x are rarely reported * Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.80.37 38 P. Kaushal et al. Cytologia 80(1) (Chatterji and Timothy 1969, Jauhar 1981). Being apomictic in mode of reproduction, gene transfer through hybridization approach is difficult. However, owing to recent knowledge on uncoupled apomixis components, fertilization of unreduced egg cell with reduced pollen (known as 2n+n or BIII hybridization) (Rutihauser 1948) is one of the possible approaches to obtain gene transfer. Uncoupling of the apomixis components (apomeiosis, parthenogenesis and functional endosperm development) has been recently reported in several agamic species (Kaushal et al. 2009). Objectives of this study were to provide evidence of BIII hybridization in P. orientale and characterization of 6x cytotype (2n=54) recovered through such uncoupling events in 4x cytotype (2n=36). Materials and methods Tetraploid accessions (2n=4x=36) of P. orientale are being maintained at Indian Grassland and Fodder Research Institute, Jhansi, India. In one of our experiments on progeny analysis of apomictic crops, self-pollinated seeds from one of these tetraploid accessions (identity P. ori 4x-2) were collected and raised in the nursery. Ploidy levels of 55 germinated seedlings were tested utilizing flow cytometric measurement of DNA contents. All the progenies were found to have 4x DNA-ploidy, except one off type plant which was confirmed to have a 6x ploidy level. This plant was separated from other siblings and was characterized for its morphological, cytological and reproductive traits, vis-à-vis its 4x progenitor (Table 1). General morphological features were observed for both the mother and the 6x progeny (given identity P. ori 6x-1) either on visual basis or metric measurements, as appropriate. Mode of Table 1. Characterization of 4x and 6x cytotypes of P. orientale. Trait (mean values) 4x cytotype (P. ori 4x-2) 6x cytotype (P. ori 6x-1) Morphological traits Plant height (vegetative) (cm) 32.2 33.5 Plant height (reproductive tillers) (cm) 74.7 91.5 Leaf length (cm) 15.7 11.5 Leaf width (cm) 0.5 0.7 Sheath length (cm) 14.5 15.2 Spike length (cm) 13.1 13.0 Number of spikelets/spikes 95.5 103 Number of tillers 200 72 Mature stigma color Purple Purple Photosynthetic pigments Chlorophyll A 12.84 10.80 Chlorophyll B 2.89 2.52 Total chlorophyll (A+B) 15.74 13.32 Pheophytin A 20.06 16.87 Pheophytin B 5.90 5.06 Total pheophytins (A+B) 25.96 21.93 Carotenoids 5.76 4.81 Total photosynthetic pigments 47.46 40.06 Reproductive traits Pollen fertility 82% 73% Mode of reproduction Apomictic Apomictic Aposporous ES (%) 100 100 Multiple ES (%) 90 74 Cytological traits Chromosome number (2n) 36 54 Average chromosome configuration 11.4II+3.3IV 22.3II+2.3IV 2C DNA content (pg) 2.83 4.25 2015 Development and Characterization of a Hexaploid Pennisetum orientale (2n=6x=54) Cytotype 39 reproduction and embryo-sac studies were performed by the methyl-salicylate mediated ovule clearing technique (Young et al. 1979). Biochemical measurements on photosynthetic pigments were also done following standard protocols. Cytological analysis on meiosis (diakinesis stage) and pollen fertility was done by staining pollen mother cells and pollen grains, respectively, at appropriate stages of development, in 2% acetocarmine. Estimation of DNA quantity was done by measuring relative DNA content using flow cytometry from leaf tissues (Kaushal et al. 2009). Results and discussion Our results showed that the 6x plant (P. ori 6x-1) had 2n=54 chromosomes exhibiting 22.3II+2.3IV average chromosome configuration (Fig. 1). The sporophytic DNA content was estimated as 4.25 pg that matched with the 6x ploidy level, as the tetraploid accession had 2n=36 chromosomes and 2.83-pg DNA content (Fig. 2). P.ori 6x-1 was perennial in life cycle similar to its 4x parent, but was less vigourous. Higher ploidy in P. orientale seemed not to affect perenniality but had modified some morphological, biochemical and reproductive attributes. Most of the metric traits were similar in both the plants except for less number of tillers and more compact spike in P. ori 6x-1 than 4x plant (Fig. 3). Both the 4x and 6x plants set seed upon self-pollination. All the photosynthetic pigments measured (chlorophylls, phaeophytins and carotenoids) were in higher amount in 4x plant than in 6x plant. Reproductive traits were also compared amongst the two cytotypes. Both of them were male fertile, though the 6x cytotype showed occurrence of sterile pollens (about 27%) (Fig. 4). Mode of reproduction, as estimated by embryo-sac (ES) studies was found to be apomictic (four nucleated aposporous ES) in both the cytotypes, though frequency of multiple ES was higher in 4x (90%) as compared to 6x (74%). In general, ES were observed to be larger in size in the 6x plant (Fig. 5). The recovery of 6x cytotype from a 4x maternal plant suggested the 2n+n (termed as BIII) Fig. 1. Pollen mother cells (PMCs) showing Meiosis I (Diakinesis) stage in 4x cytotype (upper panel) and 6x cytotype (lower panel). 40 P. Kaushal et al. Cytologia 80(1) Fig. 2. Flow cytometric histogram showing relative DNA contents from leaves of 4x (peak 1) and 6x (peak 2) cytotypes. X axis-photo-illumination, Y axis-number of events. Fig. 3. Overall morphology of plants: 4x cytotype (Fig. at left), 6x cytotype (Fig. in centre). Whole spike morphology of cytotypes (Fig. at right). Fig. 4. Pollen stainability in 4x (left) and 6x (right) cytotypes. hybridization pathway of its origin, possibly through fertilization of an unreduced egg cell (2n) with reduced male gamete (n) in the 4x cytotype (Kaushal et al. 2008). As the 4x plant was aposporous in nature, it is likely to generate unreduced egg cells in its apomeiotically developed embryo-sac (Nogler 1984). Pollen formation is normal and through reductional division in such cases, as also identified in the present study. 2015 Development and Characterization of a Hexaploid Pennisetum orientale (2n=6x=54) Cytotype 41 Fig. 5. Cleared ovule of 6x plant showing three aposporous embryo sacs (ES). e: egg cell, p: polar nuclei. Although 6x cytotypes in P. orientale have been reported earlier in limited cases as geographical collections (Jauhar 1981, Koul et al. 1999, Akiyama et al. 2011), their mode of origin was never proved. Two possible pathways have been suggested for the origin of 6x cytotypes: either through random genome duplication of 3x cytotypes (Koul et al. 1999) or by fertilization of an unreduced egg cell with a reduced male gamete (Akiyama et al. 2011). Recovery of the 6x plant from a 4x maternal plant in the present study provides the first direct experimental evidence for the later possibility. Most of the perennial polyploid grasses belonging to the Poaceae family reproduce through apomictic mode of reproduction.