Abstract: Effects of genotype and medium composition on the frequency of callus induction and green plant regeneration from anther of Nepalese rice were investigated. Cold pretreated anthers from six rice genotypes at 8±2°C for seven days were cultured on three different callus induction media designated as Callus Induction Medium (CIM 1): N6 mineral salts + N6 vitamins (2 mg L-1 each) + myoinositol (100 mg L-1) + 2,4-D (2.5 mg L-1) + KI (0.5 mg L-1) + AgNO3 (10 mg L-1) + maltose (50 g L-1), CIM 2: N6 mineral salts + MS organic salts + NAA (4 mg L-1) + Kinetin (KI) (2 mg L-1) + Silver nitrate (AgNO3) (5 mg L-1) and sucrose 60 (g L-1) and CIM 3: CIM 2 without AgNO3.. The callus induction frequency was significantly affected by rice genotypes and genotype x medium interactions. The efficiency of callus induction (calli/anther) was higher in CIM 1 (14.1%) followed by CIM 2 (12.54%) and CIM 3 (10.3%). CIM 2 was found to be superior for the recovery of green plants. Among genotypes, only the calli from Chandanath -3 and Khumal-4 were able to differentiate into green plants. In this study the calli induced on medium containing 2, 4-Dichloroacetic acid (2, 4-D) had lower regeneration ability than the medium supplied with α-naptthalene acetic acid (NAA). This study also revealed that the temperate cultivars (hill rice) were more responsive to anther culture than the tropical ones (terai rice).

Abstract: Distant hybridization in the genus Oryza is realized as an efficient Biotechnological tool for plant breeding work to introgress useful gene/s from diverse array of wild relatives into cultivated rice. This study was carried out to improve the germination frequency of hybrid embryo to enhance the crossability between O. sativa sub spp. indica and wild species. Three cultivars of indica rice viz. IR 64, Radha 4 and IR 69618 - CMS A line (cytoplasmic male sterile A line) were pollinated with the pollen of O. latifolia, O. minuta and O. officinalis. Hybrid caryopsis containing embryos were rescued at tenth day of pollination. In vitro germination frequency of rescued embryos were compared by culturing embryo alone and embryo with a bit of ovary during 2005-06 at Biotechnology Unit, Khumaltar, Lalitpur, Nepal. Culture was maintained at 25±10C under dark until germination and there after continuous light. In majority of the cross combinations, the germination frequencies were found to be higher when embryo excised and cultured with small portion of ovary. This technique yielded up to 100 per cent germination which were later employed to study the crossability between species. Depending upon the cultivars of O. sativa, the frequencies of crossability varied from 0.53 to 3.08 per cent with highest for Radha 4/O. minuta. A total of 38 hybrid plants were successfully produced from 88 cultured embryos isolated from 2644 pollinated florets. Inclusion of a bit of ovary along with embryo in in vitro culture was found to be an effective method not only to improve the germination frequency of hybrid embryo, but also to increase the crossability between cultivars of cultivated rice and their distant relatives.

Abstract: Doubled haploid regenerated through anther culture of wide cross rice hybrids is one of the potential tools to produce various useful genetic and cytogenetic stocks for molecular studies. In this study, an interspecific hybrid derived from Oryza sativa and O. rufipogon was subjected to anther culture with the aim to regenerate doubled haploid population for their characterization. A total of 21 green and 31 albino plants were obtained from 1904 anthers of O. sativa cv. Himali x O. rufipogon. Six types of androclonal variants were identified based on morphology and panicle characteristics of regenerated plants. Among 21 plants, only six were found diploid with partially fertility. Despite the sterility and preponderance of wild traits, some of the plants showed useful traits such as increased in panicle and spikelet length, reduced shattering and awning, and large size anther as in O. rufipogon. Two of the regenerants were superior for panicle related traits over either parent. The genotyping study using SSR markers also revealed the substantial variation among regenerants. At least three allelic variants were recorded among individual plants based on our preliminary study. The regenerated doubled haploid plants showed large amount of useful variation both at phenotype and molecular level and they can be exploited to enrich the current rice gene pool.

Abstract: The present study was carried out taking single F1 wheat and four maize varieties, viz. Arun-1, Arun-2, Khumal Yellow and Rampur Composite, to determine the efficiency and influence of maize genotypes on various parameters of haploid formation. Wheat spikelets were hand pollinated with freshly collected maize pollen, and 1 ml of 100 ppm 2,4-D was immediately injected on the uppermost internode. Twenty-four hours after 2,4- D injection, the cups of the florets were filled with the same solution of 2,4-D for two more consecutive days. Seventeen days after pollination, the embryos were excised and cultured in half-strength MS basal medium supplemented with 30 g/l sucrose, and 7 g/l agar. The cultured embryos were maintained at 25°C with 16/8 hours light/darkness after treating in the dark for seven days at 4°C and incubation in the dark for seven days at 25°C. Application of 2,4-D after pollination was found to be essential to the recovery of culturable size of embryos. The significant effect of maize genotypes on frequency of ovary development, embryo formation and haploid plant per pollinated floret was observed. The mean percentages of embryo formation and haploid plants per pollinated floret varied from 5.17 to 21.45 and 0.96 to 10.15, respectively, depending upon the maize varieties used. The highest frequency of embryo recovery and plant per floret was found when wheat F1 was pollinated with Arun-2 followed by Arun-1 and Khumal Yellow. It is suggested that the production of dihaploids (DHs) in wheat can be enhanced by using more responsive maize genotypes as pollinators.

Abstract: Buckwheat (Fagopyrum spp.) plays a pivotal role in sustaining the livelihood of the resource poor mountainous farmers of Nepal. The yield potential of cultivated species, however, is very low and unstable due to the complex reproductive system and self-incompatibility. The present study was carried out to standardize the in vitro breeding protocol by crossing three Nepalese species viz. F. tataricum, F. esculentum and F. cymosum to produce interspecific hybrids. Seven day old hybrid embryos were aseptically rescued and cultured on MS medium supplemented with 0.2 mg/l IAA, 2 mg/l BA, and 3% sucrose. Ten interspecific hybrids, resulting from crosses between F. tataricum x F. esculentum and F. cymosum x F. esculentum, were successfully produced. The highest crossability frequency (4%) was recorded in F. tataricum (h) x F. esculentum (t). Reciprocal differences and strong pre-fertilization barriers were recorded in some of the cross combinations. Further success in recovering interspecific hybrids needs improvement in embryo rescue technique and selection of compatible morphs.

Abstract: Determination of ploidy level and tagging of species specific useful agronomical traits in wild relatives of crop plants are of crucial value to introgress alien gene/s of interest into cultivated background through wide hybridization. Four Nepalese wild species of rice viz. Oryza nivara, O. rufipogon, O. officinalis and O. granulata were collected and grown in the green house during 2001-2002 at IAAS, Rampur, Chitwan, Nepal to assess the ploidy level of these species and characterize the species specific different phenotypic traits. Chromosome number of pollen mother cells was recorded at Diakinesis and Metaphase I by harvesting and fixing young florets at suitable stages following usual technique of chromosome preparation. Morphological and seed characteristics of each species were recorded from early vegetative stage to until harvest and after harvest, respectively, from randomly selected ten sample plants. Chromosome counting revealed that all the wild species of rice had consistent chromosome number 2n = 24. The species exhibited specific variations in the observed traits. Additionally, they had also common wild characteristics like spreading growth habit, high stigma exertion rate, high spikelet shattering, dark green leaves with purple to dusky brown basal leaf sheath colour and 2-3 branching of culm. Based on the growth habit and panicle characteristics, O. rufipogon exhibited marked ecotypic differentiation. The present study confirmed that all wild species of Nepal are essentially diploid with an array of potential traits for the improvement of cultivated rice.

Abstract: Embryo rescue is an efficient plant breeding tool to accomplish wide hybridization in the genus Oryza.Following this technique several intra and intergenomic hybrids have been successfully produced in wide arrays of crop plants. The present study was carried out taking different cultivars of O. sativa as female parent and O. officinalis and O. granulata as male parent to assess the crossability affinity between cultivated and wild species. All the F1 embryos prior to abortion (7-10 day old) were aseptically excised and cultured in vitro during 2001-2002. Culture was maintained at 25±10c under dark until germination and then continuous light (~110 foot candle). The germination of embryo among hybrids varied from 0-66.67%. Interspecific hybrids were successfully produced only from O. sativa x O. officinalis. The crossability between O. sativa and O. officinalis ranged from 0-2.44%, depending upon the cultivars of O. sativa used, whereas it was almost zero for O. sativa x O. granulata suggests that the existence of strong crossability barrier between these species. The present results showed that embryo rescue is a potential technique to overcome post fertilization barriers; however, it is no longer effective where abortion of embryo occurs at early stages of development. Therefore, efforts should be concentrated to optimize the conditions for the rescue of hybrid embryo aborting at very early stages of development.

Abstract: This study was under taken to elucidate the effect of genotypes and media compositions on callus induction from mature rice seeds. Three different callus induction media, designated as A (N6 mineral salts + N6 vitamins, 2 mg/l each + myoinositol, 100 mg/l + 2,4-D, 2.5 mg/l + kinetin, 0.5mg/l + AgNO3, 10 mg/l + maltose, 50 gm/l); B (MS organic salts + N6 mineral salts + NAA, 4 mg/l + kinetin, 2 mg/l + AgNO3, 5mg/l and sucrose, 60 gm/l); and C (B media without AgNO3), and six rice genotypes viz. Jumlimarshi, Tilki, Jethobudo, Manshara, Masuli and Pahenle were evaluated. The modified N6 medium supplemented with 2, 4-D, 2.5 mg/l and AgNO3, 10 mg/l exhibited better performance in callus induction. Among genotypes, callus induction frequency was higher (100%) in Masuli, Tilki and Jumlimarshi regardless of media tested. The positive effect of AgNO3 was only observed in medium A for quality callus induction and subsequent plant regeneration. The genotype Tilki performed better regarding plant regeneration (27.77%). Therefore, it is suggested that application of medium A is advantageous to accomplish overall efficiency of callus induction and plant regeneration from seeds of various rice genotypes.

Abstract: