CHAPTER 3 GENETIC VARIATION FOR PHOTOSYNTHETIC CAPACITY

3.3.1 Experiments

Results were organised in four experiments (Table 3.1). Experiment Aus1 was set up in a glasshouse at CSIRO Black Mountain, Canberra (-35.271875, 149.113982), from March 31st

to June 30th 2012, artificial light was used in June to extend the photoperiod to 16 h and

temperature was controlled to 25/15 °C (day/night). Two seeds were sown in cylindrical pots of 1.06 L (15 × 5cm) with 75:25 loam:vermiculite containing basal fertilizer, and one plant per pot was kept for the experiment which was organized in a randomized block design, three blocks representing each repetition for the high nitrogen treatment (+N) and other three blocks for the low nitrogen treatment (-N). Extra fertilizer Thrive (~300 mL per pot of 1.77g L-1N:27%, P:5.5%, K:9%) was applied each week for +N treatment until

83 DAE. A severe low nitrogen treatment was obtained irrigating the pots with water without fertilizer 1.5 month before measurements. Plant emergence was on April 8th. 23

days after emergence (DAE) and the flag leaf was measured at anthesis (GS58-69) from 73 to 83 DAE. The collection of plants used in this experiment is called the “early vigour set” and it was measured at anthesis so the acronym used for these data is EVA (“early vigour anthesis”); further information is described in the germplasm section. Six to seven

genotypes per day at similar plant stage with three repetitions were measured and sampled. Details of traits are in section 3.3.4.

59 Table 3.1 Details of experiments

Set of genotypes Description Key measurements

Stage Traits

Aus1 = Glasshouse experiment. CSIRO Black Mountain, Australia (2012)

EVA(-N),(+N) 16 genotypes 3 repetitions Low/high N Anthesis 73-83 DAE A, Vcmax25, J SPAD, Narea

Aus2 = Glasshouse experiment. CSIRO Black Mountain, Australia (2012)

BYPB(-N),(+N) 30 genotypes 2 repetitions Low/high N Pre-anthesis 48-56 DAE A, Vcmax25, J SPAD, Narea

Aus3 = Field experiment. GES-CSIRO, Australia (2013)

BYPB 28 genotypes 4 repetitions Pre-anthesis 46-54 DAE A, Vcmax25, J SPAD, Narea EVA 2 genotypes 4 repetitions Anthesis 62-67 DAE A, Vcmax25, J SPAD, Narea CA 21 genotypes 4 repetitions Anthesis 60-67 DAE A, Vcmax25, J SPAD, Narea

Mex = Field experiment. CENEB-CIMMYT, Mexico (2012-2013)

CB 30 genotypes 3 repetitions Pre-anthesis 67-82 DAE A, Vcmax25, Jg, Jf SPAD, Nmass CA 30 genotypes 3 repetitions Anthesis 88-103DAE A, Vcmax25, Jg, Jf SPAD, Nmass, Narea

EV= Early vigour set; BYP= BUNYIP set; C= CIMCOG set. B= before anthesis (pre-anthesis); A= anthesis. DAE= Days after emergence. See text for traits abbreviations.

Experiment Aus2 was carried out in a glasshouse at CSIRO Black Mountain, Canberra (- 35.271875, 149.113982), from October 12th to December 11th 2012, and temperature was

controlled to 25/15 °C (day/night). Three seeds were sown in pots of 5 L with 75:25 loam:vermiculite soil mix containing basal fertilizer, and two plants per pot were kept for the experiment which was organized in a randomized block design, two blocks

representing each repetition for the high nitrogen treatment (+N) and one block for the low nitrogen treatment (-N). For the +N treatments extra fertilizer Aquasol (~300 mL per pot of 1.77g L-1N:23%, P:4%, K:18%) was applied every three days from 41 days after

emergence (DAE) to 56 DAE. Treatment –N was less severe than Aus1, it was obtained irrigating the plants with water without fertilizer 10 days before measurements. Plant emergence was on October 17th and the flag leaf was measured before anthesis (GS49-57)

from 48 to 56 DAE. Measurements were done in one plant per pot for +N and in the two plants per for –N. The collection of plants used in this experiment is called BUNYIP and it was measured before anthesis so the acronym used is BYPB. Details of germplasm used

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are below in section 3.3.2. Six to ten genotypes per day at similar plant stage with two repetitions were measured and sampled.

Experiment Aus3 was carried out in the field at CSIRO Experimental Station at Ginninderra (GES), Australia (-35.199837, 149.090898) from September 25th to December 17th, 2013. The

emergence of plants was on October 4th, 2013. From 1 to 75 DAE the average maximum

from daily temperature (Figure 3.1) was 22.4 °C and the minimum 7.7 °C, in total 142 mm of rain and an accumulative thermal time of 1,126.8 °C. CA and EVA subsets of wheat genotypes were sown in the same experimental design of two randomized blocks. Each block was subdivided into 30 plots (5 × 6). Next to this experimental design, another experimental design of two randomized blocks for the BYPB collection was sown. In this case, each block was subdivided into 42 plots (7 × 6). Each plot for both experimental designs was 5 m × 1.8 m. It contained a single genotype sown in 10 rows, 18 cm apart, and approximately 200 plants m-2. Plots were fertilized and irrigated optimally in all conditions. The BYPB subset of wheat

genotypes was measured in the flag leaf before anthesis (GS40-55) from 46-54 DAE where the maximum temperature reached was 28.3 °C and the minimum 5.4 °C. When EVA (GS69, 62-67 DAE) and CA (GS56-69, 60-67 DAE) subset of wheat genotypes were measured, the maximum temperature reached was 32.2 °C and the minimum 4.3 °C. Measurements and sampling were done twice in two plots, resulting in 4 repetitions for 4 to 5 genotypes per day that were at similar plant stage. In this experiment many plants were at the same stage at the same time and fewer wheat genotypes could be measured: 2 wheat genotypes from EVA, 20 wheat genotypes and 6 triticale genotypes from BYPB, and 22 wheat genotypes from CA. They are marked with asterisk in tables 3.2 and 3.3. Details of germplasm used are in section 3.3.2.

Experiment Mex was carried out in the field at Centro Experimental Norman E. Borlaug (CENEB) research station, located in the Yaqui Valley, Sonora, Mexico (27.370837, - 109.930362) for a winter-spring cycle. The sowing was on November 23rd, 2012 and the

harvest on April 30th and May 2nd 2013 (150 and 152 DAE respectively). Plant emergence

was on 2nd, December, 2012. From the 1 to 138 DAE, the average maximum temperature

from daily temperature (Figure 3.1) was 26 °C and the minimum 8.3 °C, in total 15.38 mm of rain and an accumulative thermal time of 2,364.6 °C. Plants were organised in a

randomised 5 × 6 lattice experimental design with three repetitions. Each repetition (10 × 3 plots) enclosed two subdivisions of 5 × 3 plots. Each plot (2.4 m × 8.5 m) contained a single genotype sown in 6 rows, two beds in the middle with two rows each and two beds in the edges with one row of the same genotype, the second row in the edges corresponded

61 to the next genotype or a filling genotype. Beds followed the system 56-24, where 56 cm is the furrow width and 24 cm is the raised bed width. Plants were grown under optimal management in the field. There were in total five auxiliary irrigations, the total 500 mm of water applied. First fertilization was at soil preparation with 50 kg ha-1 of N and 50 kg ha-1

of P and a second fertilization in the first irrigation of 150 kg ha-1 of N. The CB subset of

wheat genotypes was measured in the flag leaf before anthesis (GS49-57) from 67 to 82 DAE, where the maximum temperature reached was 29.7 °C and the minimum 1.5 °C. The CA subset of wheat genotypes was measured in the flag leaf at anthesis (GS60-69) from 88 to 103 DAE, where the maximum temperature reached was 32.1 °C and the minimum 2.5 °C. Measurements and sampling were done in one plant per plot; 3 to 6 genotypes per day were measured at similar plant stage with 3 repetitions.

Figure 3.1 Meteorological conditions in Obregon, Mexico and Ginninderra, Australia. Daily observations from days after emergence (DAE) during measurements in Australia (1-75 DAE) and in Mexico (1-38 DAE). Minimum and high temperature in pink, precipitation in blue bars, minimum and maximum humidity (white circles) and solar radiation in red.

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