Data were collected for the following parameters.
5.3.3.1 Reproductive traits
a. Flower head emergence per plant: Six counts were made to note head production from 12 November 2011 to 13 February 2012. The first five counts
were taken at intervals of 10 days and heads showing petals were counted for each individual plant. This determined the starting date of flowering for all families. Last count was taken only for families used in inheritance of maturity study. In this count each family was divided into three portions i.e., the first plant, the middle portion (consisting of the 3 middle plants) and the last-plant. Heads present on the first and last plant were counted individually. For the middle portion, heads in an 18 x 18 cm quadrat were counted at 3 different places (Figure 5.3).
First plant middle portion last plant
Figure 5.3. Diagram showing locations of head count at five positions of a family. Points a, b and c show quadrat positions for the middle portion.
b. Pollen stainability: Pollen viability of 15 selected families was checked by the method adopted in experiment-1.
c. Selfing:Three heads per plant were used to observe seed setting as a result of selfing. They were bagged and gently rolled daily between thumb and finger until all the florets got deflexed.
d. Per head measurements:Mature heads were removed from each plant during the second week of February 2012. First, bagged heads for selfing were evaluated. Then open-pollinated heads of a similar age were selected most preferably from middle top part and avoiding the peripheral hanging portion of the plant. Five normal and mature heads from each end plant were taken. For the middle portion, 15 heads were taken randomly with 5 heads from each
Full count Full count b a c
plant. Data for seeds per floret, florets per heads and peduncle length were recorded and used to derive data for seeds per head, florets per plant and seeds per plant. Heads used for measuring selfing, pollen stainability, ovules count, peduncle length, florets per head, seeds per head and seeds per plant, were of the same age.
e. Per plant measurements: Data on a plant basis was derived by multiplying head production data of second last count taken on 25th of December 2010, multiplied with per head data sampled at the end of experiment i.e., during second week of February 2011. Visual scoring was done to observe the maturity of flower heads on 11thof February 2011 and 17thof March 2011.
5.3.3.2 Vegetative traits
a. Plant growth: The growth of a family was measured by its foliage dry mass and covered area. Foliage mass was harvested from plants from 22nd to 27th November 2010. Plants were trimmed back at the height of 5cm from the top edge of the pot. The height of 5cm was suitable to remove only foliage, without harming or removing the stolons. In this way the buds formed in the stolons remained safe and available for producing heads in the subsequent season. A stick showing 5cm height was fixed in the first pot and was used as a reference to trim back all plants of that family. Stolons were permitted to grow into adjacent side pots of soil. The side stolons were not removed. However, their foliage, flowers and buds were trimmed to the 5cm height. The foliage mass was oven dried at 1000C for 16 hours and its weight was used as “foliage dry weight” in data analysis. Covered area of each family was measured after the taking last counts for head production.
b. Leaf area: The areas of randomly selected healthy and fully grown leaves were measured with a leaf area meter (LI-COR Model LI-3000 Portable Area Meter). The middle lobes of five leaves per plant for the end plants and 15 leaves for the middle group were measured. Families used for evaluation of selfing performance were included in this sample. An additional 12 families were included, based on difference in their leaf sizes. In total 27 families were measured. Leaf area was represented by the single middle lobe of the leaf in cm2.
c. Stolon and nodal density: After the last head count and sampling, plants were trimmed back. During the third week of February 2011, selected families were washed with a water blaster to clean their stolons and roots. Potting mixture and leaves were washed away by the water pressure. Plants were dried in the sun. Plants of a family were separated from each other without disturbing the plant shape (especially stolons). To note stolon and node density, a 216 x 217 mm card was used with four windows cut in it according to the shape and dimensions shown in Figure 5.4. Stolons expanding and crossed half way of the window towards the side 3 and 4 were counted in each window. Any stolon heading towards or reaching side 1 and 2 was ignored. This achieved a count in that window of all stolons originating from the centre of the plant. Any stolon entering into the window from the sides was taken as not originating from the centre of the plant and was ignored. Nodes were counted on all visible stolons in all windows. The data expressed density of stolons and nodes per unit area. The stolons of each plant were removed from roots and their weight was recorded after oven drying for 24 hours at 80oC. This weight was used as “stolon dry weight” in data analysis.
Figure 5.4. Dimensions of the card with four windows used to measure density of stolons and nodes.
Window 4 Window 2 Window 1 Window 3 1 2 3 4 2 3 4 2 2 3 4 1 4 1 3 1 217 mm 216 mm 51 mm 51 mm
5.3.3.1 Root traits
The following root traits were studied for the same families which were selected for stolon data.
a. Root dry mass: Roots mass was recorded after separating the roots from stolons and oven drying them at 80oC for 24 hours.
b. Tap root diameter:Primary tap root diameter was measured at the base of the crown with an electronic Vernier Caliper (Mitutoyo digital Caliper- 500.550.551).
c. Woody tap root length: Length of intact woody portion of the tap root was measured till its terminal branched end.
5.4
ANALYSES AND RESULTS
Data from 81 families representing eight different generations were analysed (Table 5.3). Except for peduncle length and 1000 seeds weight, all data were transformed using the Box-Cox transformation in GenStat v12. During transformation, T. uniflorum data were excluded to achieve an acceptable level of normality. Thus T. uniflorum was excluded from all analyses, other than where its inclusion specifically mentioned.
Table 5.3. Details of generations and families involved in the experiment.
Serial
No. Generation
No. of
families Code of families
1 BC1F1 3 83, 84, 85 2 BC1F2 2 16, 22 3 BC1F2 (selfed) 3 18, 24, 26 4 BC1F3 14 8, 17, 27, 38, 44, 50, 52, 59, 62, 65, 75, 79, 80, 86 5 BC1F3 (selfed) 2 64, 76 6 BC2F1 2 25, 34 7 BC2F2-BI 15 20, 30, 31, 41, 43, 48, 49, 51, 54, 58, 66, 70, 71, 72, 74 8 BC2F2-IB 16 6, 11, 12, 14, 21, 28, 29, 36, 39, 40, 45, 55, 56, 57, 60, 78 9 BC2F2 (selfed) 2 7, 23 10 BC3F1 16 9, 10, 13, 15, 32, 33, 35, 37, 42, 46, 47, 61, 67, 68, 69, 73 11 white clover 2 53, 77 12 white clover (selfed) 1 19 13 T. uniflorum 3 63, 81, 82