Ales phenotype

Pre-Basic Seed: identify the Eritrean seeds

A comparison with cereals: oats, Barley, wheat, and rye in the green house

1. Local varieties with region of origin and address of the person or organization to whom enquiries about the variety should be sent.
   1. Name of the farmer Daniel Kahsay Tsighe
   2. Region and address-Duabaruwa Debub
   3. To enquire to (those who worked) Dr. Eden Tareke.
2. Communicating the name of the person(s) or organization(s) responsible for the maintenance of the variety;
   1. Name Daniel Kahsay Tsighe
3. Liaising with the maintainer of the variety;
   1. Name Daniel Kahsay Tsighe
4. Providing written agreement for the multiplication of seed outside the Country of Registration to the appropriate Designated Authority;
5. Supplying an official description of the variety to be multiplied/tested

   1. Description of the seed which year/Season/specification/generation of the plant tested

   1. Designation of Categories of Seed

      The following categories of seed are recognized in the Scheme:
      i. Pre-Basic Seed

   2. Pre-Basic Seed

      Pre-basic seed is defined a seed of generations preceding Basic seed and may be at any generation between the parental material and the Basic seed.

      - Controlled/ maintained by. Daniel Kahsay Tsighe (farmer)

      - Subject to compulsory pre-control test.

      - Cannot be commercialized and it has to be used for further multiplication.

      - Produced officially by the recognized Institute/organization.

      Samples of Eritrean grain (Ales), barley, wheat, oats and rye were grown in five0.5-liter pots with standard soil under controlled atmosphere in a greenhouse so that the plants can be closely examined and critically compared throughout the period of growth.

      Every week in the early stage and every other week in the late stage of growth the plats were inspected. The inspections were made in a standardised way cf. approaches I-IV below (as much as possible to work within the limits of reasonable resources) in order to achieve results of sufficient accuracy and reliability.

6. Authenticating the identity of the seed to be multiplied.

   Seed crop is inspection with regards to:

   A. Phenotypes compared in a in greenhouse experiment

   B. In field conditions and characteristics

   C. Nutrient (protein, amino acid, dietary fibers (soluble and insoluble) content

A. Inspection and phenotypes compared in a in green house experiment

This report describes the different comparisons performed to closely investigate any differences in the phenotypic appearance between the Eritrean cereal grains locally known as (Ales) and the registered cultivars of what, oats, barley and rye.

Plants were grown in 0.5-liter pots with standard soil, in a greenhouse under halogen lamps, giving a photon flux density of 240 μmol/m2/sec with a photo-period of 18 h. Day and night temperatures were 25°C and 16°C respectively.

Approaches and Results

1. Germination

   Seeds (4/pot) Ales along with oat, barley, wheat and rye were planted on- 03/09/2014 in in 0.5-liter pots with standard soil, in a greenhouse. The day of germination was recorded and compared.

   Results: All seeds, except wheat which started to germinate on 6th day, germinated and showed the first meristem on the 4th day after sowing See Figure 1.

   Figure 1. The pots were the seeds were seeded and the seeding conditions on day 4

2. Nature of the first meristem

   The nature of the first meristem was for the cereals was observed and compared to see the similarities and potential differences that may distinguish Ales from the other cereals.

   Results: Ales and rye showed brown color pattern in the whole meristem which spanned from top to bottom of the entire meristem distinguishing them from the other cereals.

   The color was slightly pink in rye meristem which disappeared later stage of growth. Other cereals showed typical greenish and white color from top to bottom. See Figure 2.

   Figure 2. Picture of the first merstm of rye, Eritrean grain, Barley and Swedish oats.

3. Nature of second leaflet

   The phenotypic appearances of second leaflets were inspected for all the cereals.

   Results: In Ales and rye the second leaflet emerged from the main stem, which was intact. While in the other cereals, wheat, barley and oats, the second leaflet emerged from first main shoot but it was kept deep inside of first leaf let (looks like every leaflet have only one origin which is deep inside the first leaf sheath).

   Figure 3. Nature of second leaflet in wheat and Ales.

4. Nature of the direction of leaf

   The twisting nature of each leaflet, whether clock wise or anti clock wise was inspected and compared

   Results: Clockwise twisting direction of each leaflet was similar in all the cereals gain subjected for the study except barley Figure 4.

   Figure 4. The twisting nature of the leas in Ales /Eritrean gran, barley and wheat.

5. Nature of shoot

   The nature of the stem, leaf width and color in all the cereals were inspected and compared to Ales.

   Results: In the Eritrean cereal grain the main stem is intact and growing as such. In addition, new leaf sheath is coming from main stems in distinct petiole. Area of leaf sheath is relatively small but was connected with main stem at a definite place. However, in Rye, the main stem is not intact and sheath is wide and coming from the bottom line. Another difference between Ales and rye was that the brown color persisted in Ales throughout while in rye it disappeared after two weeks.

   Ales takes less space per plant because the tillering which is maximum 3-4/plant and that the shoot is thinner compared to the cereals mentioned above. More over since each tiller is intact like the main shoot and produces healthy seeds and the cereal has short growing period.( 2.5-3 months) the yield/area and /time is enhanced.

   Figure 5. The shoot appearance in barley rye and Ales.

6. Flowering

   The time for flowering was recorded and compared for all the cereals grown in the green house.

   Results: Flowering time was shorter (28th-30th day) for Ales compared to flowering time for the other cereals (40th -42nd day).

   Figure 6. The picture showing the flowering stages and the nature of spike of Eritrean seed.

7. Spikelet arrangements and nature of the hear in the grain

   Nature of panicle and the arrangement of each spikelet in single panicle is different for cereals. All the spikelet are tightly packed and have a unique pattern which was keeping in a straight way of packing in the pedicel. For barley and wheat each spikelet have own stem like appearances which is attached to the pedicel and arranged in an irregular way (from 4 corners for wheat).

   Nature of hair in Ales was similar to that of rye, what and oats. But interesting difference in hair length in each spikelet was observed. The hair length in barley was 15cm, in Ales, 10-11 cm in wheat and oats 8-9cm. The hair in the spikelet of Ales was much thinner that in the others.

B. Phenotypic characters inn field conditions and characteristics

1. 100 kernel weight??
2. Yield per hectare??
3. Plot size- (seed/area) ??
4. Frequency of seeds/panicle??
5. Harvesting time
6. Duration of growth, flowering, and seedling
7. Characteristics of Shoot (stem, petiole, pedicel, branching)
8. Characteristics of seed (hull, groat, endosperm, embryo)

C. Nutrient (protein, amino acid, soluble fiber) content

1. Introduction

   The nutritional content of Ales was analyzed with respect to protein, amino acid, total and soluble fiber Values.

   Protein content on average in the analysed Ales seeds Table 1, and protein levels analyzed in seeds from three generations Ales grown in in the field in Dubarwa year 2011, 2012, 2013 Table 2, protein content in Ales grown in greenhouse three generations Table 3, and protein levels in Ales grown in different regions in Eritrea: Dubarwa, Emnihayli, Embaderho, Emnitselim, Laquen and Adiwatot was determined Figure 7. The amino acid levels in all the grains from the different regions was also determined and compared to amino acid levels in in soybean, pea, and oats Table 4. In addition dietary fibers both soluble and insoluble were determined and presented in Table 5. In Table 6 comparison is made between protein content in Ales and oat varieties selected for their high protein content grown under the same conditions.

2. Methods
   1. Total protein analysis

      Total protein content was determined by measuring total nitrogen content using Elemental Flash A 1112N (Thermo Fischer scientists, Delt, Netherland) and a modified Dumas method (Dumas J, B 1831). 50mg (5-8 seeds) of KHT and KNHT oats were combusted in a sealed furnace combined with a thermal conductivity detector. The total protein percentage was calculated by measuring the total nitrogen content in seed flour assuming a protein to nitrogen conversion factor 6.25. Aspartic acid was used as the standard.

   2. Determination of total amino acid by Acid hydrolysis method

      Amino acid analysis of defatted and non-defatted samples of both KHT and KNHT oats were performed on Biochrom 30 series analyzer (Stenberg, M et al 2002, Trachur R, 1965, Fermin B.C, 2003). The samples were hydrolyzed with 5ml of 6M HCl with 0.1% phenol in hydrolyzing tubes (in triplicate). The tubes were sealed and placed in a rotating rack in the oven at 1100c for about 20-24 hours. The samples were placed in centrifuge tubes and centrifuged at 4000 rpm for 10 minutes at room temperature. 2ml supernatant was transferred in to a round bottom flask and 100 µl of Norleucin internal standard was added. The entire samples were evaporated by putting in an evaporator working under vacuum at 40 0c temperature until the time to get it dry. 5ml of 0.2M lithium citrate buffer at pH (2.2) was added to the dry sample and mixed for 3-4 minutes in an ultrasonic bath. 1.5ml was then transferred to an eppendorf tube and centrifuged for 5 minutes at 13000 rpm. This supernatant was then filtered through 0.45 micron polytetraq fluroethylene filter, collected and analyzed

   3. Total dietary fiber analysis (total and soluble fiber)

      Total dietary fiber analyses were carried out by using the principal of the method developed by Asp et al 1983. A series of enzymes such as pepsin NF (200mg, 2000 FIP-U/g, Merck, Darmstadt, West Germany ), pancreatin 4xNF ( 200mg, activity equivalent to 8 x USP specifications, Sigma, St. Louis, MO) and Termamyl 60L (Novo A/S, Copenhagen, Denmark) were used for the digestion of protein and starch. All incubation steps were carried out in ordinary Erlenmeyer flasks. Samples were agitated horizontally in special water bath according to the specified time and temperature. Duplicate samples are run routinely.

      Weighed oat samples (1 g) were transferred to a 0.5L Erlenmeyer flasks with a magnetic flea in it. Cold 0.1M Sodium phosphate buffer at a pH 6.0 were added to the sample. For the digestion of starch 100µl of Termamyl enzymes was added. Samples were incubated subsequently for 15 minutes in a boiling water bath and allowed to cool for 10 minutes. After mixed thoroughly for 5-10 minutes, 0.2N HCL was added and the pH was adjusted to 1.5 with 5m HCL solution. The pH electrode was rinsed with distilled water in to the sample. Incubation of samples for 60 minutes at 40 oC were performed in a shaking water bath after adding 1 ml of pepsin in the sample. After 1 hour of this horizontal agitation pH was adjusted as 6.8 with 5N NaOH. The pH electrode was rinsed with distilled water in to the sample. One ml of pancreatin was added to the entire mixture and incubated for 1 hour at 40 oC in a water bath with shaking speed of 4.5 rpm. Finally 4-5ml of 0.5NHCL was added to the sample and the pH was adjusted accurately as 4.5 with 0.2 NHCL.

      Filtrations of soluble and insoluble fibers were carried out separately by using a modified Fibertec M equipment (Tecator AB). Soluble and insoluble fibers were collected individually in weighed crucibles contain known amount of celite. Crucibles with porosity 2 (pore size 40-90 µm) contain 500-520mg of acid washed and incinerated celite (Kebo Grave, Stockholm, Sweden) were used as the filtration aid.

3. Results
   1. Protein content in Ales seed acquired from Dubarwa

      Wt% protein in defatted oat flour
      Sample Ales (n=12 from,5 regions)	Nitrogen 22 ± 0 .2	Amino acid 20.3 ± 3.8

      Table 1. Average Protein content in Ales seed seed
   2. Protein content Ales (3 generation cultivated in Dubarwa year 2011-2014)

      Date of harvesting	Generation	Protein content
      2011	1st	22,3%
      2013	2nd	22,7%
      2014	3rd	23,7%

      Table 2.
   3. Protein content Ales (3 generation cultivated in green house)

      Date of harvesting	Generation	Protein content
      11.05.2013	1st	25%
      14.08.2013	2nd	24%
      06.05.2014	3rd	23,6%

      Table 3.
   4. Protein levels measured in Ales collected from different regions in Eritrea

      Figure 7.

   5. Amino acid levels in Ales compared to amino acid levels in soybean, pea, and oats in mg/g protein

      Amino acids	Ales	soybean	pea	Oats
      Asp	19.3	4.80	2.15	1.7
      Thr	9.4	45.00	29.16	15.22
      Ser	11.8	20.20	8.37	5.64
      Glu	94.1	27.90	10.99	7.18
      Pro	30.8	60.30	44.69	40.87
      Gly	13.5	24.40	8.60	8.04
      Ala	13.2	30.90	10.04	8.38
      Cys		23.00	8.84	8.55
      Val	15.7	6.20	0.00	2.74
      Met		27.60	9.08	9.06
      Ile	12.2	6.40	4.78	4.28
      Leu	21.4	20.00	7.17	6.67
      Tyr	8.1	28.30	15.77	12.65
      Phe	15.9	20.40	5.98	5.30
      Lys	10.5	22.00	9.80	9.06
      His	7.3	23.30	16.25	7.18
      Arg	15.7	21.60	5.74	3.76
      % protein	25.6	37.10	21.51	11.80
      Amino acid (recovered)	286.3	449.40	218.92	166.38

      Table 4.
   6. Dietary fiber analysis

      Reference value (mean)	Insoluble fibre in %	Soluble fibre in %	Total fibre in %
      Reference value oats	8	3.1	11.1
      Oat	6.1	2.8	9.6
      Ales	8.96	3.71	12.67

      Table 5. Total dietary fiber (soluble and insoluble) analysis if Eritrean seed
   7. Comparison of protein and dietary fiber comparison of Ales and other top ranking cereals
   8. Evaluation of protein content of high protein oat lines (wild varieties) and Ales grain grown under the same conditions.

      Sr. No	Name of at lines	% of protein
      1	Lodi	16%
      2	Dal	17,9%
      3	Goodland	20,4%1
      4	Ales	25%

      Table 6. Comparison of protein content in Ales to known high protein variety cereals