El Plazo Razonable en sede Prejurisdiccional

Combined GISH and FISH experiments were carried out, using Cy3-dCTP labelled pTr5S and Fluor-X-dCTP labelled genomic DNA from T. ambiguum as probes, on the mitotic metaphase chromosome preparations of the selected BAR09 and BAR10 hybrids. The procedure for hybridization, post hybridization stringent washing and counter staining with 4’, 6-diaminodino-2-phenylindole (DAPI) was followed as described by Ansari et al. (1999) with some minor modifications. For this experiment, already screened slides that were a few days old (two slides per genotype) with comparatively high mitotic index were used. They were treated with 0.1 ug/ul RNase in 2x SSC for 55 minutes at 37OC in a humid chamber. Thereafter, the slides were washed three times for one, five, & four minutes respectively in 2x SSC, fixed in 4% paraformaldehyde for 10 minutes at room temperature and dehydrated through a series of alcohol (ethanol) concentrations followed by air drying. Before hybridization, chromatin denaturation was carried out at 72oC in 70% formamide-2xSSC (0.3 M sodium chloride, 0.03 M sodium citrate) for two minutes and the material quickly dehydrated through an ice-cold ethanol series (70%, 90% & 100%) and then air dried for 10-20 minutes at room temperature.

A 20 ul aliquot of hybridization mixture containing 2.8 and 0.91 ng/ul of each probe (genomic DNA of T. ambiguum and 5S rRNA), 50 % formamide, 20% dextran sulphate, 0.5% SDS in 2x SSC and 10 and 40 times excess of sheared unlabelled DNA of T.

occidentale and T. repens respectively, was heat denatured for 10 minutes at 90 OC. After chilling on ice for a few minutes the mixture was applied to an already marked area on each slide and then coverslips made from polythene disposable waste bags were added. One slide from each hybrid underwent another denaturation in a thermocycler (Techne PHC-3) for two minutes at 72OC after which the temperature gradually came down to 37 OC and then the hybridization was allowed to go on at 37 OC for 30 minutes before the slides were shifted to a humid chamber. The other slide from each hybrid was transferred directly to the humid chamber set at 37 OC without putting in the thermocycler for a second denaturation. The hybridization was carried out overnight (almost 20 hours) in the humid chamber at 37 OC. Next morning the cover slips were removed carefully from the slides and then post hybridization washing was done at 420 C

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using two changes of 2xSSC for 5 minutes, one change of 50 % (v/v) formamide in 2xSSC for 10 minutes and then again two changes of 2xSSC for 5 minutes. After cooling of the jar for 10 minutes, slides were rinsed in 2xSSC and 4xSSC/Tween 20 at room temperature. The chromosomes were counterstained in 4’,6-diaminodino-2- phenylindole (DAPI) and then mounted in Vectashield (Vector Laboratories, USA) under a glass coverslip. Slides were then studied under a Nikon Microphot-SA epifluorescence microscope. The images were taken using an AxioCam MRm CCD camera (Carl Zeiss, Germany) attached to the microscope and processed with ISIS imaging software (MetaSystems, Germany). The individual photographs were later customized for best contrast and brightness by using Adobe Photoshop software.

The procedures for hybridization, post hybridization stringent washing and counter staining with DAPI in GISH/FISH on mieotic chromosome spreads followed the protocol of Ansari et al. (1999) except for one additional step of pepsin treatment. After RNase treatment, the slides were incubated in 10 uM HCl at room temperature. Thereafter, 180 ul of working solution of pepsin (5ug of pepsin/ul of 10 Mm of HCL) was added to each slide and covered with a plastic coverslip. Slides were incubated at 37OC for 30 minutes in the humid chamber so as to digest and remove the proteinaceous background caused by the cytoplasm density of PMCs. After pepsin treatment, the slides were twice washed with 2xSSC involving two changes of 2xSSC for five and four minutes respectively. The rest of the process was carried out as given for GISH/FISH on mitotic chromosome preparations.

At least 40 PMCs were analysed from each selected BAR hybrid to examine, in particular, the behaviour of T. ambiguum-derived chromosomes.

3.11 Morphological characterization of BAR09 and BAR10 hybrids

For morphological characterization of BAR09 and BAR10 hybrids in the field, experiments containing selected progeny plants along with the original BAR hybrids and genetically colour marked white clovers (as controls) were planted on 5-12 May, 2010 and 06-08 June, 2011. For these experiments, clonal cuttings were obtained from the plants selected to be included in the experiment. Care was taken that the cuttings were of the same size and physical condition. Each cutting consisted of 3-4 cm of stem with one active growing point and one main root with three fully opened trifoliolate leaves. Initially these were planted in plastic pots of 8 cm diameter filled with 1:1 sand and peat

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potting mix and placed to establish in a glasshouse with natural day length and a temperature range of 15-25 OC. The pots were watered as needed and a complete soluble fertilizer (Yates Thrive®) was applied fortnightly to maintain soil fertility. After one month they were shifted to a sandpit with a depth of 45 cm where the experiment was laid out in a randomized complete block design (RCBD) with four replicates. Each plant was allocated an area of 0.36 m2. They were watered daily, usually in the evening, and fed with a complete soluble commercially available plant nutrient supplement (Yates Thrive®) on a weekly basis at the rate of 250 ml per plant. Morphological observations on experiments containing BAR09 hybrids were recorded on the above-ground qualitative and quantitative traits in January, 2011. During May, 2011, this experiment was harvested destructively by digging up the plants and data were recorded on above and under-ground components of the plants. Similarly, data on different morphological characteristics of BAR10 progenies were recorded during January and February, 2012. Following destructive harvest in January, 2012, plant components were oven-dried at 80 O

C for 48 hours and then the dry weights of above-ground parts, root system and total biomass were recorded using an electronic scale. The traits on which data were collected are given below:

Stem type (whether stoloniferous or not) (SToNST), i.e. rooting at the nodes or not Stolon number per plant (SN)

Average stolon length based on five randomly selected stolons (cm) (ASL)

Stem anchorage on the scale of 0-10 (0=no nodal rooting at all, 10 nodal rooting from every node as in white clover)

Number of inflorescences per plant (NI)

Growth habit, whether determinate or indeterminate (GH) Florets/inflorescence (head) (F/H)

Average peduncle length based on five randomly selected peduncles (cm) (APL) Leaflet shape (length/width ratio) (LS)

Average petiole length (cm) (APL) Average stolon thickness (cm) (ST)

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Flowering, whether axillary, terminal or both (FATC) Seeds/Inflorescence (head) (S/H)

Main root thickness immediately under soil surface (cm) (MRT)

Main nodal root thickness adjacent to the point of attachment (cm) (MNRT) Above ground dry weight (gm) (AGDW)

Root dry weight (gm) (RDW) Total biomass (gm) (TBM)

Root weight % of the total biomass (RW%TBM)

3.12 Analysis of variance, ANOVA

Analysis of variance (ANOVA) was carried out by using Genstat 12th edition (Payne et

al., 2010).

Table 3.1 Original 4x hybrids with genomic formula RRAO used in strategy-1 based on using