The project was approved late July 2008 and activities have started as expected for the postrainy season 2008-09. We could not have a meeting with all project partners before the start of the season. However, we had a couple of strategic meeting with partners based in India (ICRISAT, NRCS, ILRI), in early September to ensure that planned activities for the Rabi season would proceed as planned in the project proposal. Essentially, we met to decide on locations and material to include in testing.
Field activities have been performed as planned. Details are described in the different project activities below. The kick-off meeting with all partners of the project has been held between 23-25 March 2009 at ICRISAT. This meeting was originally planned for January but was postponed because of the aftermath of the Bombay events. Minutes and program of the meeting have been circulated and are available upon request.
The project is well on track. Detailed analysis of data gathered so far and only recently collected needs to be further pursued. The use of the lysimetric system to assess pre-post anthesis water use, a key component of information that this project aims to produce, appears to fulfil really well the expectations and thorough analysis of data now needs to be done. Excerpts of these analyses and progresses made thus far are provided in what follows.
Activity 1.1. Complete the marker-assisted backcross development of single/multiple stay-green QTL (stgA, stgB, stg1, stg2, stg3, stg4) NILs in elite parents including sweet sorghum S35.
These materials were advanced prior to the 2008-09 trial and seeds were multiplied and dispatched to NRCS for multilocation trials reported below (Activity 1.3)
Activity 1.2. Test single/multiple stay-green QTL for stay-green expression in the field and grain/stover yield/quality at Patancheru under terminal drought conditions.
The single and multiple stay-green QTL introgression lines in two different genetic backgrounds (R16 and S35) were evaluated in two moisture regimes (irrigated and stress) during postrainy season of year 2008-2009 at two sites in India, Patancheru and Tandur . The stay-green QTL isolines in R16 genetic background were evaluated in a replicated 42-entry (including parents and checks) trial in an alpha-design with 4-row plots (of 6m length). The stay-green QTL isolines in S35 genetic background were accommodated in a replicated 56-entry trial with a field design similar to R16 genetic background trial. The trials were sown during second week of October 2008. The observations on Green Leaf Area (starting from 2 weeks before flowering till maturity) and other related traits on agronomic performance of these QTL isolines were recorded. The whole plant straw samples were harvested on plot basis, and grinding of these samples will be completed by second week of June 2009, for stover fodder quality traits. On the basis of results from this trial, best single QTL isolines in both genetic backgrounds will be identified for development of double QTL introgression lines.
Activity 1.3. Test selected single/multiple QTL isolines in multi-location trials of India
These test were performed at several locations by NRCS. During Rabi season (October-April 2009), two sets of material, 42 lines of R 16 based and 56 lines of S35 based genetic background stay green QTL introgression lines were evaluated for postflowering drought tolerance. The trials were conducted at three locations, Tandur (Andhra Pradesh), Rahuri (Maharashtra) and Bijapur (Karnataka) in 6x7 (R16 based genetic material) and 7x8 (S35 based genetic material) alpha designs with three replications. The sowing was done on Oct 3, Oct 17 and Oct 27, 2008 at Tandur, Bijapur and Rahuri, respectively. Two water regimes were imposed by withholding the irrigation during the post flowering period. Unfortunately, the trials conducted at Rahiri and Bijapur were abandoned before flowering due to severe shoot fly attack due to late sowing. Excess rains during first weak of October resulted in the flooding of fields, which did not allow us to prepare the field for sowing in time.
At Tandur, trial on the evaluation of S35 based genetic material was successfully conducted and the required data set were recorded. Following observations were recorded.
Plant stand
Days to 50% percent flowering and grain physiological maturity,
Plant height at Flowering and physiological maturity
Green leaf area estimation of top six leaves from flowering onward
Panicle number and dry weight,
Stover and grain dry weight
100-grain mass
Stover Quality parameters- stover samples for quality analysis was handed over to ILRI.
As data analysis is yet to be completed, the results from this trial will be reported in the next report.
Activity 2.1. Model analysis of increased WUE (pre/post anthesis water use) stay-green under different climatic scenarios for both Australia and post-rainy season India
Soil depth measurements have been taken to feed into APSIM. The work on APSIM will fully start once the post doctoral fellow is recruited, which should take place towards the beginning of Year 2 of the project.
Activity 2.2. Assess variation for WUE/WU in the GCP-genotyped sorghum reference collection and in single/multiple stay-green QTL isolines from 1.1.
This work was carried out at ICRISAT
Evaluation of the full reference collection of sorghum for transpiration efficiency under well-watered and water-stressed conditions, and for the rate of water loss per unit of leaf area
We assessed all the accessions (375 + 3 controls) of the reference collection for transpiration efficiency (TE). The trial was planted on October 27th and the TE experiment was carried out between 8 and 27th December 2008. Plants were grown in large pots (11" diameters, containing 11 kg of Alfisol). Four seeds were planted in each pot, later on thinned to one seedling per pot at 2 weeks after sowing. Twelve plants (pots) were grown for each entry. Plants were grown under fully irrigated conditions until 6 weeks after sowing. At that stage, 4 replicated plants per genotype were harvested to evaluate plant biomass before imposing the treatment in the other 8 plants, which were used for the stress (water stress, WS) and control treatment (well-watered, WW), that is 4 replicated plants for each treatment. For that, the remaining plants were saturated with water. Since there were 378 entries in the trial, only one replication within each treatment could be handled per day and therefore the treatment imposition was staggered over 4 days. On each day, one replication within each treatment (WS and WW) were saturated with water, bagged with plastic bag around the stem the following morning, and weighed immediately after. Bagging prevented soil evaporation. Same procedure was followed for the 3 other replications. The procedure for each of the treatment was as follows:
1. Drought stress set: WS plants were exposed to progressive water stress by letting plant loose no more than 150 g per day during the first 4 days after imposing stress, no more than 100 g per day in the following 4 days, and no more than 75 g per days during the rest of the experiment. Since pots were weighed every 4 days only, this corresponded to a maximum water loss of 600 g over 4 days, 400 g in the subsequent 4 days, and then 300 g for any 4 days interval in the rest of the experiment.
2. Well-watered set: WW plants were maintained well-watered by re-adjusting pot weight close to field capacity on those days when the pots were weighed (every 4 days), and by adding water two days after weighing to bring back pots close to field capacity, based on transpiration data (from previous weighing intervals).
All plants were harvested when the transpiration of the drought set fell below 10% of the transpiration of the well-watered set. Harvest and transpiration data have been performed but data entry has still to be completed and should be reported during our next meeting.
Evaluation of a portion of the reference set (with relatively similar flowering time during Rabi) and selected stay-green QTL introgression lines for water uptake under stressed conditions in lysimeters (2.0-m long and 25-cm diameter tubes), and for the proportion of water used prior/after anthesis
A set of 210 entries, including 152 accessions from the reference collection + 58 marker-aided stay-green QTL introgressed lines in the background of S35 and R16 were assessed in large and long PVC cylinders (2.0 m long and 25 cm diameter), mimicking roughly the soil volume that sorghum plants would have at usual field planting densities. The trial has been sown on 20th Oct 08. Re-saturation of the soil profile was performed between Nov 13 and 15th, by adding 3 liters of water to each cylinder to ensure that all lysimeters were saturated at the time of imposing the stress. Immediately, after saturation, polyethylene bead were applied to the soil surface to prevent soil evaporation, at a rate of 600 mL, giving a layer of approximately 1.2 cm, sufficient to limit soil evaporation by about 80%. First weighing of the cylinders was taken on 19-20-21-22 November 08. In each case, one full pit was taken, corresponding to one and half replications in each of the treatment (these are 630 cylinders, which is the maximum that can be weighed accurately in a single day). Subsequently, weighing was done following the same sequence, so that the interval between weighing was the same for all treatments and replications. Two treatments were applied: a water stress treatment and a fully irrigated control. Procedure for each of these treatment was as follows:
1. Water stress treatment: No water was added from the time of saturation, i.e. 15th Nov 08, except 1000 mL applied during grain filling on each of 02nd and 09th Jan 09. A total of five weighing were taken for the water stress treatment, i.e. 21-22 Nov, 5-6 Dec, 19-20 Dec, 7-8 Jan, 19-20. Plants were physiologically mature at the time of the last weighing and thus were harvested soon after weighing. Plant parts were separated in leaf, stem, panicle and grain weights.
The data showed a range of variation in water extraction between 10193 g plant-1 to 15260 g plant-1, i.e. about 50% variation in water extraction between the highest and lowest water "extracters".
One of the hypothesis of the project is that staygreen could be a consequence of differences in the pattern of water use, with staygreen material likely to be those using a relatively lower proportion of water during the pre-anthesis period, to favor more water availability for grain filling. We found a clear negative relationship across the 210 entries, between the amount of water taken before anthesis and that taken after anthesis, showing that genotypes taking up more water before anthesis took up less water after anthesis. How this relates to grain yield still needs to be analyzed.
The PVC experiment gave also the opportunity to assess TE across a fairly large range of genotypes, over a long period of time (from 4 weeks after sowing until maturity). TE value varied between 2.44 g kg-1 water transpired to 6.09 g kg-1 water transpired, and so showed a very large range of variation. A detailed analysis is need to investigate how differences in TE relate to the presence or absence of certain staygreen QTL. In short, in S35 background, few staygreen QTL introgression lines had TE above S35. Introgression lines with Stg 3, Stg4 or Stg B had all fairly high TE equal or superior to S35. In R16 background, most Stg B, and Stg3 and few Stg4 introgression lines had high TE, equal or superior to R16. In any case, there were entries from the sorghum reference collection with TE higher that the highest of the staygreen trial entries.
2. Well-watered treatment: Six cylinder weighing have been done on: 19-20 Nov 08, 3-4 Dec 08, 17-18 Dec 08, 5-6 Jan 09, 21-22 Jan 09, and 2-3 February. Plants were harvested soon after the last weighing. For genotypes maturing earlier, the head had been harvested before to avoid grain losses. However, all the stover biomass was harvested at same time. Analysis of the water extracted has been done until after the final weighing of the samples.
Total water use under well-watered conditions varied between about 10.5 kg to about 42.3 kg of water per plants from 4 weeks after sowing until maturity. The highest and lowest water users under well-watered conditions are reported below. Biomass and grain data have been performed and reveal a large range of variation. As for the DS treatment, the trial was an opportunity to assess TE over a long period of time (from 4 weeks after sowing until maturity) in a large set of entries. There also, the very interesting thing is that data revealed also a good range of variation in TE. Thorough data analysis is still needed and is planned for the next meeting in Australia during the National Conference on Sorghum (7-8 August 2009).
Activity 2.3. Conduct an ex-ante analysis of the economic benefits (increase in grain/fodder quantity/quality) of incorporating stay-green and/or improved pre/post anthesis water uses/WUE traits in existing sorghum varieties in Australia and post rainy season India
Monthly sorghum stover sampling from six sorghum stover traders in Hyderabad was taken up November 2008 to provide new baseline data for stover quality - price relationships. From November 08 to March 09, the average price per kg dry stover was 12.7 cents. For comparison from November 2004 to March 2005 the average stover price was 6.7 cents.

The project is fully underway. For the second postrainy season of the project, the trials in ICRISAT and at the partner locations were carried out almost rain-free, which allowed an excellent expression of the stay-green phenotype and an excellent discrimination of the stay-green introgression lines. From the six different QTL that have been introgressed from donor parent B35, it is fairly evident that StgA, StgB and Stg3 all contribute to a clear expression of the stay-green phenotype and some of the lines contribute to enhanced seed yield and biomass under terminal drought.

Interestingly, the 2009-10 season confirms the value of several StgA, StgB and Stg3 introgression lines across locations on trials at the partner's stations. Due to this excellent proof of concept of the value of several stay-green QTL, discussions during the most recent planning meeting centered on: (i) developing a di-allele scheme to combine beneficial QTLs; and (ii) choosing six popular varieties from Rabi sorghum (North Karnataka, West Andhra Pradesh, Maharashtra) to initiate the introgression of beneficial QTLs into these varieties.

The lysimetric facility used to evaluate a number of traits related to plant water use and water extraction has revealed a wealth of variation in the germplasm for transpiration efficiency (over 50% range of variation) and a range of variation for water extraction from 12 to 15 litres per plant. This provides new sources of variation for critical traits that can be fed into breeding programs. Interestingly, certain stay-green QTL enhance either transpiration efficiency or water extraction, although this depends on the genetic background where they are introgressed. For instance, Stg1 and Stg2 enhance water uptake in an S35 background (which has limited capacity to extract water), while there is no QTL that improves water extraction in an R16 background, which is already endowed with high water extraction capacity. Therefore, stay-green QTL can be dissected for the different traits they control at the gene level. However, a prior knowledge of these traits in target cultivars for introgression is critical.

The postdoctoral fellow has been recruited and is at present undertaking training in APSIM in Queensland, Australia. Initial work will consist of mapping yields across Rabi sorghum states in India and extrapolating soil conditions prevailing at each test site. This mapping will then be used to test the effects of specific QTL on yield across a range of locations.

The project is still in full swing. For the third post-rainy season of the project, trials have been repeated at ICRISAT and at three partner locations during 2010-11, using a subset of the most promising entries tested in the 2009-10 season, i.e. 18 entries in S35 and 18 entries in R16 backgrounds. Harvest of the trials has been completed and fodder quality assessment is in progress. These same entries have also been tested in the lysimetric system to evaluate precisely the pattern of water extraction and its relation to yield and leaf development parameters. Some of these entries have also been tested in pot studies in a controlled environment facility to evaluate traits related to the plant's control of water use under well-watered and water stressed conditions. The introgression of stay-green QTLs into six popular varieties from the Rabi sorghum main track (North Karnataka, western Andhra Pradesh, Maharashtra) is in progress. Characterization of the Rabi sorghum environment has been done to elicit the type of stress that is most common in order to better target the choice of traits needed for adaptation. This has involved the collection of climatic data from Rabi sorghum districts and productivity data from these same districts, as well a thorough APSIM simulation. In a nutshell, Rabi sorghum primarily undergoes flowering and post-flowering terminal drought, which is fully in line with the project's objectives. Efforts have been made and are still on-going in Year 3 to deliver publications, with one paper accepted in Functional Plant Biology, and one paper currently under review (Crop and Pasture Science). A meeting has also recently been held to discuss other potential publications in view of the value of the data collected since the beginning of the project. We have agreed to have one manuscript prepared to report the result of the 44 introgression line entries (+ checks) in the S35 background and one from the 31 introgression line entries (+ checks) in the R16 background, across the DSR and ICRISAT locations using data for the period 2009-10. This manuscript will be drafted by Dr HS Talwar, Principal Scientist at DSR. A second manuscript will report the detailed yield trials carried out at ICRISAT and will relate expression of the stay-green phenotype and its relationship with yield, using the 2009-10 data and possibly the 2008-09 data for the S35 background. This manuscript will be drafted by Dr Santosh Deshpande, and will report, in particular, the clear expression of the stay-green phenotype in StgA, StgB and Stg3 introgression lines. A third manuscript, headed by Dr Michael Blummel, will report on the assessment of fodder quality data in different locations and under different water regimes at each location. An ex-ante assessment of the value of introgression of stay-green QTLs has been executed and a fourth manuscript is coming up. A meeting was held in Queensland in April 2011 to update project partners on progress. We have also discussed the possibility of a Phase 2 for this project, capitalizing on the great progress achieved in Phase 1. An outline of the ideas that have been exchanged will be sent to the ACIAR program manager as the basis for an initial dialogue.