Volume 8, Number 7, July 2014 (Serial Number 75)

4. Summary

For the evaluation of the joint environmental impact, the dendroindication method is used. The spruce reference reaction in 20 years (from 1989 to 2008) due to environmental changes is expressed by the cumulative and current additional increment, which depends on the location of the stand and its morphometric characteristics. The Multiple Linear Regression model is developed, which describes the size of the environmental impact, as the arguments using indices inherent to all spruce stands of Kurzeme (stand age, average height, average diameter of the stand, the height above sea level and site index class). The unexplained information by the model expresses the specific impact value of each sampling plot. This category is represented by the effect of (local) factors specific only for the stand of each sampling plot (management regime, hydrological regime, soil characteristics, the location in the area, industrial pollution of the air, etc.). During the research planning, there was assumed that the essential impact of the polluted air comes from the industrial facilities located near Mažeikiai, Liep āja and Brocēni. The analysis of

the specific impact of the environment of each sampling plot allows a conclusion about a significant impact of the Mažeikiai Oil Refinery on the productivity decline of spruce stands of the vicinity in the period from 1989 to 2008.

References

[1] Council Directive 1999/30/EC of 22 April 1999. “Relating to Limit Values for Sulfur Dioxide, Nitrogen Dioxide and Oxides of Nitrogen, Particulate Matter and Lead in Ambient Air.” Official Journal of the European Communities L163: 41-60. [2] Лиепа И, Я. 1980. Динамика древесных ресурсов. Прогнозирование и экология (in Russian) (Stand Volume Dynamics. Forecasting and Ecology). Рига: Зинатне.

[3] Schweingruber, F. 1996. Tree Rings and Environment Dendroecology. Berne: Paul Haupt Verlag. [4] BALTI Group 2012. Latvia's Forests During 20 Years of

Independence. BALTI Group.

[5] NGO 2012. Forest Sector in Latvia. Za ļās mājas: NGO. [6] Sokal, R. R., and Rohlf, F. J. 1995. Biometry: The

Principles and Practice of Statistics in Biological Research. 3 rd edition. New York: W. H. Freeman and Company.

[7] Balmori, A. 2009. “Electromagnetic Pollution from Phone Masts. Effects on Wildlife.” Pathophysiology (16): 191-199.

[8] Balodis, V., Br ūmelis, G., Kalviškis, K., Nikodemus, O., Tjarve, D., and Znoti ņa, V. 1996. “Does the Skrunda Radiolocation Station Diminish the Radial Growth of Pine Trees?” The Science of the Total Environment 180 (1): 57-64.

[9] Liepa, I. 1996. Pieauguma M ācība (in Latvian) (Forest Increment Science). Jelgava: LLU. [10] Антанайтис, В., and Загреев, В. 1981. Прирост леса (in Russian) (Forest Increment). Москва: Лесная промышленность.

[11] Bi čevskis, M. 2005. “Egļu Astoņzobu Mizgrauzis, tā Izrais ītie Bojājumi un Ierobežošanas Metodes (in Latvian) (Spruce Bark Beetle, the Resulting Damage and Containment Methods).” R īga: Latvijas valsts meži.

[12] Bi čevskis M., 2006. Egļu Astoņzobu Mizgrauža Savairošan ās Ierobežošanas Sekmju Novērtējums AS LVM Vald ījumā Esošajos Mežos Pēc 2005. g. Janvāra

V ētras (in Latvian) (Spruce Bark Beetle (Ips typographus L.) Limiting Progress Assessment in Stock Company LVM Forests After the 2005th January Storms). Salaspils:

Latvijas Valsts Mežzin ātnes Institūts “Silava”.

July 2014, Vol. 8, No. 7, pp. 582-592

Journal of Life Sciences, ISSN 1934-7391, USA

DAVID PUBLISHING

On-farm Evaluation of Effect of Organic and Mineral Fertilizers on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa

1 2 Hide Omae 3 , A. K. Saidou and Satoshi Tobita

1. Tropical Agriculture Research Front (TARF), Japan International Research Center for Agricultural Sciences (JIRCAS), 1091-1 Maezato-Kawarabaru, Ishigaki, Okinawa 907-0002, Japan 2. National Institute of Agronomic Research of Niger (INRAN), Niamey, PO Box 429, Niger 3. Crop, Livestock and Environment Division, Japan International Research Center for Agricultural Sciences (JIRCAS), Tsukuba,

Ibaraki 305-8686, Japan

Received: April 18, 2014 / Accepted: July 14, 2014 / Published: July 31, 2014.

Abstract: To determine the best combinations of organic and mineral fertilizer applications under resource-limited conditions in the Sahel, West Africa, on-farm experiments for two years in the Fakara region of western Niger is conducted. Ten treatments were tested; six of them received one of three organic fertilizers (none, millet husks, or manure), with or without mineral fertilizer; two grew millet/(dual-purpose) cowpea intercrops with mineral fertilizer in 1:1 or 4:4 row arrangement; one grew a millet/ (spreading) cowpea intercrop with manure; and one grew a millet/hibiscus intercrop with millet husks. Manure increased total biomass by 127%-147% (P < 0.001) and millet yield by 130%-184% (P < 0.01). Mineral fertilizer increased total biomass by 27% (P < 0.01) and millet yield by 24% (P < 0.05) in 2008. The N balance was greatest and positive in the manure treatment (P < 0.001), followed by millet husks. Mineral fertilizer on the intercropped dual-purpose cowpea increased the agronomic N use efficiency of millet by 329%-483% (P < 0.01) compared with manure. The use of small quantities of mineral fertilizer on the intercropped dual-purpose cowpea, therefore, is a best combination for limited N-resources-farmers. Single manure, millet husks plus mineral fertilizer, or year-alternative-application are also recommendable depends on farmers accessibility to the resources.

Key Words: Millet, cowpea, intercrop, manure, mineral fertilizer, millet husks, sandy soil, nitrogen use efficiency.

1. Introduction  land productivity and soil fertility [3].

Pearl millet (Pennisetum glaucum), an important The soil in the Sahel, West Africa, is sandy, with a food crop in the rainfed Sahelian environment, low nutrient-holding capacity and low levels of P, N produces a moderately reliable grain yield [4]. Millet [1], and organic C. Additional environmental is traditionally cropped with cowpea (Vigna constraints, notably high temperatures and low annual unguiculata). Both crops are sown at very low rainfall (300-800 mm), force farmers to practice densities (< 5000 hills/ha) with no fertilizer [5]. low-input forms of agriculture such as shifting Although improved millet cultivars have been cultivation [2]. Recent decades have seen large developed, farmers still grow local landraces, which population increases, the breakdown of traditional provide grain for humans, fodder for livestock, and shifting cultivation systems, and a rapid decline of stalks for fencing, precluding their use as organic

fertilizer. Small doses of mineral fertilizer increased Corresponding author: Hide Omae, Ph.D., Senior

production [6, 7], but farmers cannot afford to buy researcher, research fields: agronomy, plant physiology, soil

fertility. E-mail: homae@affrc.go.jp. mineral fertilizer, and thus low-cost inputs must be

On-farm Evaluation of Effect of Organic and Mineral Fertilizers 583

on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa

found. Suitable alternatives include intercropping the biomass and yield of millet/cowpea intercrops, and and cereal-legume (or non-legume) rotation at

(2) to compare the best combination of organic and increased planting density, and improved cultivars of

mineral fertilizers with low-cost technologies such as cowpea [5, 8-14]. Different row arrangements in

the use of an improved cowpea cultivar with an cereal-legume intercrops also have also been optimum row arrangement in millet/cowpea and compared [15-17].

millet/non-legume intercropping. Among fertilizers, mineral fertilizer [18], manure

2. Materials and Methods

[19, 20], and both [21] have been tested. Mineral fertilizer appears to be the best means of restoring the

An on-farm experiments was conducted during the nutrient balance, increasing crop yields, and raising

2008 and 2009 cropping seasons in four villages rural incomes [3]. The addition of manure would

(Bokossay: 13°25.13  N, 2°47.27 E; Yerimadey: allow assessment of the dynamics of C and organic N

13°28.65  N, 2°42.25 E; Kodey: 13°23.50 N, and P in the evaluation of sustainable local 2°49.23  E; Mourey Koala Zeno: 13°35.02 N, agroecosystems [22].

2°38.78  E) in the Fakara commune, Dantiandou The objectives of this study were (1) to study the

district, Tillaberi prefecture, western Niger, about 50 effect of low-cost organic and mineral fertilizers on

km northeast of Niamey, the capital (Fig. 1).

Fig. 1 Location of the experimental plots.

On-farm Evaluation of Effect of Organic and Mineral Fertilizers

on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa

The experimental plots were treated as mother weeding, respectively with micro-dosing technique [6, fields of mother-baby trials [23]. There were 61

7]. Each experiment used a local millet cultivar, households in Maourey Kouara Zeno, 90 in Yerima

“Haïni tchirey” (120 days to harvest). The experiments Dey, 51 in Bokossay, and 105 in Ko Dey (2009, pers.

were conducted in a strip-plot design with four comm.). The Zarma are the principal ethnic group of

replications (one per village). Table 1 shows details. this region. They are agriculturalists engaged mainly

The millet was sown at 1.0 m × 1.0 m, and thinned in rainfed production of millet (Pennisetum glaucum

to three plants per hill after 2 weeks. It was planted in (L.) R. Br.) and cowpea (Vigna unguiculata (L.)

June and harvested in October each year. Cowpea was Walp.). The prevailing soil type in the region is

sown at 1.0 m × 1.0 m. It was planted in July and Psammentic Paleutalfs, with a high sand fraction and

harvested in October each year. At physiological typical characteristics of an infertile soil [24, 25]. The

maturity, millet plants in each plot were harvested and rainfall starts from June until September, with a total

partitioned into ears and stalks. After drying, the ears rainfall of about 550 mm, which peaks in August [26].

were threshed and the total biomass was determined. From 2001 to 2007, the annual average rainfall at

Cowpea plants were also harvested at physiological Kodey was 435 mm. In 2008, the annual rainfall was

maturity, partitioned into fodders and pods. After 442.2 mm within 38 days of rain; in 2009, it was

drying, the pods were threshed. All aboveground crop 515.6 mm within 45 days of rain. The mean annual

residues were removed from the plots at end of each temperature ranged from 22.8 °C to 31.2 °C in 2008,

cropping season.

from 23 °C to 36 °C in 2009. Soil samples were taken from the top 15 cm, In 2008, in each mother field in each village, ten

air-dried, crushed, and sieved (2-mm mesh) for plots (20 m × 20 m) were established. Six of them

chemical analysis. Following wet digestion with received one of six combined fertilizer treatments:

salicylic acid-thiosulfate, the total N content was three forms of organic fertilizer (none, millet husks, or

measured at 660 nm with a Technicon Auto-Analyzer manure), with or without mineral fertilizer, and grew a

II (Pulse Instrumentation Ltd., Saskatoon, SK, Canada) millet/cowpea intercrop, using a local cowpea [28]. After extraction with Bray No. 1 solution, landrace.

available P was measured by the molybdenum blue Two of them grew millet/cowpea intercrops in 1:1

method [29]. Organic C content was determined by or 4:4 row arrangements, using a dual-purpose,

the Walkley-Black method [30]. medium-maturity cowpea line (85 days), TN256-87 [2,

The air-dry plant samples were ground 3 months 27], with mineral fertilizer. One grew a spreading

after the harvest; 200 mg of each sample was digested cowpea line, IT99-213-11-1 [2], with manure. And

with salicylic acid-thiosulfate, and the total N content one grew a millet/hibiscus (Hibiscus sabdariffa,

was measured at 660 nm with the Technicon ‘Wankoye’) intercrop, with millet husks. In 2009, the

Auto-Analyzer II. N uptake was determined by ten plots were each divided into two (9.5 m × 20 m);

multiplying the N concentration by the plant biomass, half of each was fertilized as in 2008, and the other

and the N balance was calculated as atmospheric N half was not fertilized. Millet received 9 t/ha of cow

minus N uptake. The ratio of atmospheric N in dung manure, 6 t/ha of millet husks which were

cowpea was taken from Yakubu et al. [31]. derived from refining process of millet head. For

The agronomic nitrogen uptake efficiency (ANUPE, application of mineral fertilizer, 3 g of compound

kg of aboveground biomass/kg of N applied) and the

agronomic nitrogen use efficiency (ANUE, kg of of urea per hill were applied at sowing and at first

mineral fertilizer (N: P 2 O 5 :K 2 O = 15:15:15), and 2 g

grain/kg of N applied) were determined as [32]:

On-farm Evaluation of Effect of Organic and Mineral Fertilizers 585

on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa

Table 1 Combination of treatment in experimental plots, Fakara, western Niger.

Application Intercrop

Cowpea or

Ro

history Millet/Cowpea

arrangement

Application

Hibiscus cultivar

- Millet/Cowpea

local 1:1

No

2008 Millet/Cowpea

local 1:1

Manure

2008 Millet/Cowpea

local 1:1

Millet husks

2008 Millet/Cowpea

local 1:1

Manure + Mineral fertilizer

2008 Millet/Cowpea

local 1:1

Millet husks + Mineral fertilizer

2008 Millet/Cowpea

local 1:1

Mineral fertilizer

2008 Millet/Cowpea

TN256-87

Mineral fertilizer

2008 Millet/Cowpea

TN256-87

Mineral fertilizer

2008 Millet/Hibiscus

IT99-213-11-1 1:1

Manure

2008 Millet/Cowpea

Wankoye

Millet husks

2008 & 09 Millet/Cowpea

2008 & 09 Millet/Cowpea

local

Millet husks

2008 & 09 Millet/Cowpea

local

Manure + Mineral fertilizer

2008 & 09 Millet/Cowpea

local

Millet husks + Mineral fertilizer

2008 & 09 Millet/Cowpea

local

Mineral fertilizer

2008 & 09 Millet/Cowpea

TN256-87

Mineral fertilizer

2008 & 09 Millet/Cowpea

TN256-87

Mineral fertilizer

2008 & 09 Millet/Hibiscus

IT99-213-11-1

Millet husks

ANUPE = (UB N − UB 0 )/F N 135%-152% (P < 0.001), cowpea biomass by ANUE = (Y N −Y 0 )/F N 91%-111% (P < 0.01), and millet grain yield by

where UB N is aboveground biomass of N-fertilized 130%-184% (P < 0.01). Mineral fertilizer increased plot (kg/ha), UB 0 is aboveground biomass of total biomass by 27% (P < 0.01), millet biomass by control plot (kg/ha), F N is amount of fertilizer 34% (P < 0.01), cowpea biomass by 14% (P < 0.05), N (kg/ha applied), Y N is grain yield of N-fertilized

and millet grain yield by 24% (P < 0.05) in 2008. plot (kg/ha), and Y 0 is grain yield of control plot

Repeat application of fertilizers in 2009 increased (kg/ha).

total biomass by 28% (P < 0.05) and millet biomass The results were tested by analysis of variance

by 31% (P < 0.05) compared to the application only in (ANOVA) followed by student’s t-test in JMP version

2008. Interactions among fertilizers and application

9.0.0 software (SAS Institute, Cary, NC, USA). history were not significant except for that between organic and inorganic fertilizers on millet grain yield

3. Results and Discussion

in 2008 (P < 0.05). Relative to the control, the ratio of

3.1 Effect of Organic and Mineral Fertilizer millet biomass to total biomass was higher in manure application in 2009 (P < 0.05) and in mineral fertilizer With organic fertilizer, the total (millet + cowpea),

application in both years (P < 0.05). The differences millet, and cowpea biomass and the millet grain yield

in biomass among fertilizer treatments were due to decreased in the order of manure > millet husks >

nutrient availability [33].

control in both 2008 and 2009 (Table 2). Relative to As a result of greater N input and uptake, the N the control, manure increased total biomass by

balance was greatest and positive in the manure

127%-147% (P < 0.001), millet biomass by treatment (P < 0.001), followed by millet husks (Table

On-farm Evaluation of Effect of Organic and Mineral Fertilizers

on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa

Table 2 Effect of organic and in-organic fertilizer and application history on biomass and grain of millet / cowpea intercrop at experimental fields, Fakara, Niger in 2008 and 2009.

Millet biomass ratio Treatment

Biomass (kg/ha)

Grain (kg/ha)

Millet + Cowpea

Cowpea to total biomass

62 50 0.63 0.92a Organic matter

(O) Millet husks

88 31 0.60 0.84b Non

72 28 0.65 0.87b Mineral fertilizer

69 30 0.68a 0.89a (MF)

76 42 0.59b 0.87b Application

32 - 0.90 history (AH)

Applied in 2008-09

42 - 0.86 O

Applied in 2008

n.s. n.s. * MF

n.s. ** *

n.s. n.s. n.s. O X MF

AH n.s.

n.s. n.s. n.s. O X AH

n.s. n.s. n.s. MF X AH

n.s. n.s. n.s. O X MF X AH

n.s. n.s. n.s. *, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).

Table 3 Effect of organic and in-organic fertilizer and application history on N balance in millet / cowpea intercropping at experimental fields, Fakara, Niger in 2008 and 2009.

N balance N uptake / N input Treatment

N input by the

N input from

application

atmosphere

N uptake by millet

N uptake by cowpea

(kg N/ha)

(kg N/ha)

(kg N/ha)

(kg N/ha)

(kg N/ha)

32.8a 0.4b 6.3 Organic matter

(O) Millet husks

8.1b 0.6b 6.2 Non

-7.2c 1.6a 6.3 Mineral fertilizer

1.9 -7.4c

14.0a 0.7b 6.6 (MF)

10.8b 1.0a 6.0 Application

25.2 2.0 38.8 39.7a 0.8 1.6b history (AH)

Applied in 2008-09

59.5 59.5 17.4 1.4 12.9 19.2a

25.2 2.5 38.8 -15.2b 0.8 10.9a O

Applied in 2008

59.5 0.0 17.4 1.7 12.9 14.5b

*** *** n.s. MF

* ** n.s.

*** n.s. *** O X MF

n.s. *** n.s. O X AH

*** n.s. ** MF X AH

** n.s. * O X MF X AH

n.s. n.s. n.s. *, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).

3). There was a significant difference between organic 9% compared to millet husks (P < 0.01). Total soil N application and no application in N uptake/input in

and P contents increased between years (P < 0.001). 2008. Repeat application in 2009 increased N input

Manure made the N balance positive. Lack of and uptake by millet by 32% (P < 0.05), and resulted

nutrient availability makes it negative [33]. There is in a bigger positive N balance and a smaller N

much evidence that N and P availability sets crop uptake/input ratio. Use of mineral fertilizer increased

yields in West Africa [34], but past experience shows N input and uptake by cowpea, which contributed to

that their supply to plants should be organically increase N balance by 9% (P < 0.05) and lower N

mediated [35]. In sandy soils, it thus appears uptake/input ratio by 30% (P < 0.01) in 2008.

fundamental to manage all components that affect soil Manure increased total soil N by 11% (P < 0.05,

fertility. Biological processes are crucial to sustaining Table 4). No application increased soil P content by

the fertility of sandy soils, as they control N flux [36].

On-farm Evaluation of Effect of Organic and Mineral Fertilizers 587

on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa

Table 4 Effect of organic and in-organic fertilizer and application history on total soil N, Bray I-P and organic C in millet / cowpea intercrop at experimental fields, Fakara, Niger in 2008 and 2009.

Total N

Bray I-P

Organic C

Treatment

(mg/kg)

(mg/kg)

Organic matter

Millet husks

fertilizer (MF)

Application Applied in 2008-09

history (AH)

Applied in 2008

Year (Y)

AH n.s.

*, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).

As in other pedoclimatic zones, the assessment of N nitrogen use efficiency (hereafter, ANUE) of millet by and P in agricultural systems on tropical sandy soils is

90%-288% (n.s.) compared with organic fertilizers

a useful tool for defining sustainable intensification (manure and millet husks), followed by organic plus plans necessary to respond to population increases and

mineral fertilizers (Table 5). Millet husks plus mineral global change issues [37, 38].

fertilizer increased ANUPE of total biomass by 117%, Mineral fertilizer increased agronomic nitrogen

that of millet biomass by 107%, that of cowpea uptake efficiency (hereafter, ANUPE) of total (millet

biomass by 177%, and ANUE of millet grain by 171% and cowpea) biomass by 107%-213% (n.s.), that of

compared with millet husks alone, although the millet biomass by 142%-273% (n.s.), and agronomic

differences were not significant. In contrast, manure

On-farm Evaluation of Effect of Organic and Mineral Fertilizers

on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa

Table 5 Agronomic N uptak e and N use efficiency in different application of organic and in-organic fertilizer at experimental fields, Fak ara, Niger in 2008 and 2009.

ANUE (kg/N kg) Treatment

ANUPE (kg/N kg)

Millet Cowpea

20.7 17.6 3.1 2.4 0.3 Organic / In-

Millet husks

organic Manure + Mineral fertilizer 33.5 27.9 5.7 4.1 0.0 fertilizer (O)

Millet husks + Mineral fertilizer 45.0 36.4 8.6 6.5 -0.2

9.3 -1.5 Application

Mineral fertilizer

64.8 65.4 -0.6

Applied in 2008, 2009 31.3 28.4 2.9 6.0 -0.2 history (AH)

Applied in 2008

n.s. n.s.

n.s. n.s. O X AH

AH n.s.

n.s.

n.s.

n.s. n.s. *, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).

plus mineral fertilizer did not increase either ANUPE fertilizer in 2009 (Table 6). Relative to millet husks, or ANUE compared with manure alone.

manure also increased total biomass by 67%-120% (P The use of crop residues plus fertilizer quadrupled

< 0.001), millet biomass by 69%-176% (P < 0.001), grain yield relative to the control, and the use of

millet yield by 106%-231% (P < 0.001) in 2009, and fertilizer doubled millet yield relative to the control.

cowpea yield by 74%-1550% (P < 0.01) in 2008. Crop residues can significantly improve the efficiency

When compared within same fertilizer applications, of use of the applied fertilizer [39]. The differences

mineral fertilizer on intercropped cowpea line between manure and millet husks when mineral

TN256-87 in the 4:4 row arrangement reduced the fertilizer is added might be due to differences in N

millet biomass ratio to total biomass in 2009 (P < mineralization.

0.001) relative to 1:1 row arrangement. Manure on the The single application of fertilizers in 2008

intercropped cowpea line IT99K-213-11-1 reduced increased ANUPE of the total biomass by 45%,

cowpea yield by 89% (P < 0.01) compared to that on increased that of millet biomass by 42%, and

the intercropped local cowpea in 2008. Millet husks increased that of cowpea biomass by 72%, but

on intercropped hibiscus reduced the millet biomass decreased ANUE of millet by 20%, although no

ratio compared to that on intercropped local cowpea differences were significant.

(P < 0.01). Repeat application of fertilizers increased total biomass by 31% (P < 0.05), millet biomass by

3.2 Combination Effect of Intercrop, Cowpea Cultivar 35% (P < 0.05), millet yield by 38% (P < 0.05), and and Row Arrangement increased millet biomass ratio (P < 0.05) compared to

With organic fertilizer, manure increased total

single application.

biomass by 53%-133% (P < 0.001), millet biomass by Different combinations of millet and cowpea 52%-173% (P < 0.001), and millet yield by cultivars gave different yield performances [9], and 77%-183% (P < 0.001) compared with mineral

the selection of genotypes with a suitable combination

On-farm Evaluation of Effect of Organic and Mineral Fertilizers 589

on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa

Table 6 Effect of intercrop, cowpea cultivar, row arrangement and application on biomass and grain of millet / cowpea intercrop at experimental fields, Fakara, Niger in 2008 and 2009

Biomass (kg/ha)

Grain (kg/ha)

Cowpea or Millet biomass ratio Combination of treatments (C)

Hibiscus to total biomass Intercrop

Millet + Cowpea or

Millet

Cowpea or

2008 2009 2008 2009 Millet / Cowpea

Cowpea or Row Hibiscus cultivar arrangement

88ab 34 0.65 0.92a Millet / Cowpea

TN256-87

Mineral fertilizer

88ab 65 0.71 0.79b Millet / Cowpea

TN256-87

Mineral fertilizer

64ab 21 0.71 0.91a Millet / Cowpea IT99K-213-11-1

local

Mineral fertilizer

6c 12 0.55 0.92a Millet / Cowpea

57b 55 0.66 0.92a Millet / Hibiscus

- 89 0.60 0.76b Millet / Cowpea

Wankoye

Millet husks

Millet husks

99a 33 0.57 0.87a

- 44 - 0.89a Application history (AH)

Applied in 2008-09

Applied in 2008

- 45 - 0.85b

C n.s.

** n.s. n.s. ***

n.s. n.s. n.s. * C X AH

AH n.s.

n.s. n.s. n.s. n.s. *, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).

Table 7 Agronomic N uptake and N use efficiency in different combination of intercrop, cowpea cultivar, row, organic and in-organic fertilizer application at experimental fields, Fakara, Niger in 2008 and 2009.

Combination of treatment (C)

ANUPE (kg/N kg)

ANUE (kg/N kg)

Cowpea Intercrop

Millet +

Cowpea

Millet or Hibiscus cultivar arrangement

Cowpea or

Row

Application

Cowpea or

Millet

or

Hibiscus Millet / Cowpea

Hibiscus

Hibiscus

13.8 21.0a 0.5 Millet / Cowpea

TN256-87

Mineral fertilizer

107.1a

93.3a

13.7 15.3ab 2.2 Millet / Cowpea

TN256-87

Mineral fertilizer

70.5ab

56.8abc

9.3bc -1.5 Millet / Cowpea IT99K-213-11-1

local

Mineral fertilizer

8.6 3.6c -0.7 Millet / Cowpea

4.2 4.9c 0.0 Millet / Hibiscus

5.4 0.6c 1.2 Millet / Cowpea

Wankoye

Millet husks

Millet husks

37.8 32.9 4.9 8.1 0.2 Application history (AH)

Applied in 2008, 2009

Applied in 2008

** n.s.

AH n.s.

n.s.

n.s.

n.s. n.s.

n.s. n.s. *, **, *** indicate significantly different at 0.05, 0.01 and 0.001 level, respectively while n.s. indicates no significantly different. Different alphabets indicate statistically significant at 0.05 level (by Student t test).

of traits with a strong relationship with and a direct space, supporting photosynthesis, despite at before the effect on yield at an appropriate density increased the

risk of insect attack.

productivity of cowpea [40]. Oso et al. [13] studied Relative to manure, mineral fertilizer on the two row spacing arrangements (2:3 and 1:1 maize to

intercropped cowpea line TN256-87 in the 1:1 row cowpea) and concluded that the planting pattern in

arrangement increased ANUPE of the total biomass intercropping appears to influence leaf infestation by

by 242%-263% (P < 0.01), increased that of millet beetles. Nambiar et al. [41] ascribed the effect of the

biomass by 246%-346% (P < 0.01), and increased intercropping system to the shading of legumes by the

ANUE of millet by 329%-483% (P < 0.01, Table 7). cereal and the consequent decrease in photosynthesis.

Relative to millet husks, mineral fertilizer on the The 4:4 row arrangement may allow cowpea more

intercropped cowpea line TN256-87 increased

On-farm Evaluation of Effect of Organic and Mineral Fertilizers

on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa

ANUPE of the total biomass by 417%-676% (P <

the global scale.

0.01), that of millet biomass by 430%-1011% (P < As conclusion, the use of small quantities of 0.01), and increased ANUE of millet by 775%-3400%

mineral fertilizer, and its combination with the (P < 0.01). There were no significantly differences

intercropped dual-purpose cowpea gave the best observed when compared within the same fertilizer

performance on ANUPE and ANUE. Therefore, this applications. Single fertilizer application in 2008

can be recommendable for poor N resources farmers increased ANUPE of the total biomass by 52% (n.s.),

like in the Sahel, West Africa. In addition, single that of millet biomass by 48% (n.s.), and that of

application of manure, millet husks plus mineral cowpea biomass by 78% (n.s.).

fertilizer, or year-alternative-application of fertilizer Variation in N uptake and in N use efficiency

are also recommendable depends on the farmers depends on N fertilization [42]. The variation in N

accessibility to the resources.

uptake was higher at low N than at high N ANUE in

References

wheat decreased as N increased (to 120 kg N/ha) [32]. In our study, the application of mineral fertilizer gave

[1] Buerkert, A., Bationo, A., and Piepho, H. P. 2001. “Efficient Phosphorus Application Strategies for

higher ANUPE than manure and millet husks (Table Increased Crop Production in Sub-Saharan West Africa.”

7), maybe because of differences in N level between

Field Crops Research 72: 1-15.

organic and mineral fertilizers (mineral fertilizer, 13.7 [2] Matsunaga, R., Singh, B. B., Adamou, M., Tobita, S., kg N/ha; millet husks, 41.4 kg N/ha; manure, 109.8 kg

Hayashi, K., and Kamidohzono, A. 2008. “Yield Performance, Nitrogen and Phosphorus Acquisition of

N/ha). However, when the difference in N content Cowpea Germplasm Accessions in Low-fertile Sandy

between manure and millet husks is considered, not Soils in the Sahelian Zone.” Tropical Agriculture and only total N but also the mineralization process should

Development 52 (2): 50-57.

be related to NUE. [3] Bationo, A., Lompo, F., and Koala, S. 1998. “Research on Nutrient Flows and Balances in West Africa:

Classical breeding could improve NUE [42]. State-of-the-Art Agriculture.” Ecosystems and

Cowpea line TN256-87 is an example of breeding for

Environment 71: 19-35.

NUE, and benefits further when grown in the [4] Pandey, R. K., Maranville, J. W., and Bako, Y. 2001. optimum row arrangement.

“Nitrogen Fertilizer Response and Use Efficiency for Three Cereal Crops in Niger.” Communications in Soil

At the global scale, cereal yields and fertilizer N Science and Plant Analysis 32 (9 & 10): 1456-1482. consumption have increased almost linearly during the

[5] Subbarao, G. V., Renard, C., Payne, W. A., and Bationo, past 40 years and are highly correlated with one

A. 2000. “Long-term Effects of Tillage, Phosphorus another [43]. Under this situation, interventions to

Fertilization and Crop Rotation on Pearl Millet-cowpea Productivity in the West African Sahel.” Experimental

increase NUE and reduce N losses to the environment

Agriculture 36: 243-264.

must be accomplished at the farm or field scale. [6] ICRISAT 2001. Grey to Green Revolution. ICRISAT Farmers in the Sahel rely heavily on millet and

Annual Report, Patancheru, Andhra, Pradesh, India. cowpea. Dugue [44] estimated that in Burkina Faso,

[7] Tabo, R., Bationo, A., Diallo Maimouna, K., Hassane, O., and Koala, S. 2006. Fertilizer Micro-dosing for the

only 10% of crop residues are left in the field, despite Prosperity of Small-scale Farmers in the Sahel: Final

the known benefits to soil chemical and physical Report. Global Theme on Agroecosystems Report No. 23, properties [45]. Better combination of organic matters,

Niamey, Niger, 2006.

mineral fertilizer and N-efficient-crops, thus, should [8] Bationo, A., and Ntare, B. R., 2000. “Rotation and Nitrogen Fertilizer Effects on Pearl Millet, Cowpea and

be well considered to improve and sustain crop Groundnut Yield and Soil Chemical Properties in a Sandy

productivity under the limited and competed N Soil in the Semi-arid Tropics, West Africa.” Journal of resources situations not only in the Sahel but also at

Agricultural Science 134: 277-284.

On-farm Evaluation of Effect of Organic and Mineral Fertilizers 591

on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa

[9] Ntare, B. R. 1989. “Evaluation of Cowpea Cultivars for Agricultural Science 134: 277-284. Intercropping with Pearl Millet in the Sahelian Zone of

[22] Woomer, P. L., Palm, C. A., Qureshi, J. N., and West Africa.” Field Crops Research 20: 31-40.

Kotto-Same, J. 1998. “Carbon Sequestration and Organic [10] Ntare, B. R. 1990. “Intercropping Morphologically

Resource Management in African Smallholder Different Cowpeas with Pearl Millet in A Short Season

Agriculture.” In Management of Carbon Sequestration in Environment in the Sahel.” Experimental Agriculture 26:

Soil, edited by Lal, R., Kimbe, J. M., and Stewart, B., A. 41-47.

Raton: CRC Press.

[11] Ntare, B. R., and Williams, J. H. 1992. “Response of [23] Snapp, S., S. 1999. “Mother and Baby Trials: A novel Cowpea Cultivars to Planting Pattern and Date of Sowing

Trial Design Being Tried out in Malawi.” Target in Intercrops with Pearl Millet in Niger.” Experimental

Newsletter of the Southern Africa Soil Fertility Network Agriculture 28: 41-48.

[12] Ofori, F., and Stern, W. R. 1987. “Cereal-legume [24] Hiernaux, P., and Ayantunde, A. 2004. The Fakara: A Intercropping Systems.” Advances in Agronomy 41:

Semi-arid Agro-ecosystem under Stress. Report of 41-90.

research activities, first phase (July 2002-June 2004) of [13] Oso, A. A., and Falade, M. J. 2010. “Effects of Variety

the DMP-GEF Program, International Livestock and Spatial Arrangement on Pest Incidence, Damage and

Research Institute.

Subsequent Yield of Cowpea in a Cowpea/Maize [25] Oudwater, N., and Martin, A. 2003. “Methods and Issues Intercrop.” World Journal of Agricultural Sciences 6 (3):

in Exploring Local Knowledge of Soils.” Geoderma 111 274-276.

(3-4): 387-401.

[14] Reddy, K. C., Visser, P., and Buckner, P. 1992. “Pearl [26] FAO 2004. Global Information and Early Warning Millet and Cowpea Yields in Sole and Intercrop Systems,

System on Food and Agriculture: Sahel Weather and and Their After-effects on Soil and Crop Productivity.”

Crop Situation Report. FAO/GIEWS Sahel Report No. 2. Field Crops Research 28: 315-326.

[27] Saidou, A. K. 2005. “Variability within cowpea genotype [15] El-Sarag, E. I. 2013. “Cowpea-sorghum Mixtures as

to applied phosphorus and residual effect on sorghum Affected by Water Stress Levels and Forage Mixing

production on P deficient soil of Sudan and Sahel Ratio in Semi-arid Regions.” World Journal of

Savannas.” Ph. D. thesis, Ahmadu Bello University. Agricultural Sciences 9 (4): 325-334.

[28] Bemner, J. M., Mulvaney, C. S., 1982. “Nitrogen-total.” [16] Mohammed, I. B., Olufajo, O. O., Singh, B. B.,

In Methods of Soil Analysis. Part 2: Chemical and Oluwasemire, K. O., and Chiezey, U. F. 2008.

Microbial Properties, edited by Page, A. L., Miller, R. H., “Productivity of Millet/Cowpea Intercrop as Affected by

and Keeney, D. R. Madison Wisconsin, USA: American Cowpea Genotype and Row Arrangement.” Word

Society of Agronomy.

Journal of Agricultural Sciences 4 (S): 818-824. [29] Olsen, S. R., and Sommers, L. E. 1982. “Phosphorus.” In [17] Willey, R. W. 1979. “Intercropping—Its Importance and

Methods of Soil Analysis. Part 2: Chemical and Research Needs. Part 2. Agronomy and Research

Microbial Properties, edited by Page, A. L., Miller, R. H., Approaches.” Commonwealth Bureau of Pastures and

and Keeney, D. R. Madison Wisconsin, USA: American Field Crops 32 (2): 73-85.

Society of Agronomy.

[18] Bationo, A., Christianson, C. B., and Baethgen, W. E. [30] Nelson, D. W., and Sommers, L. E. 1982. “Total Carbon, 1990. “Plant Density and Nitrogen Fertilizer Effects on

Organic Carbon, and Organic Matter.” In Methods of Soil Pearl Millet Production in Niger.” Agronomy Journal 82:

Analysis. Part 2: Chemical and Microbial Properties, 290-295.

edited by Page, A. L., Miller, R. H., and Keeney, D. R. [19] Djiteye, M. A., Penning de Vries, F. W. T. 1982. La

Madison Wisconsin, USA: American Society of Productivité des Pâturages Sahéliens: Une Étude des

Agronomy.

Sols, des Végétations et de L’exploitation de Cette [31] Yakubu, H. J., Kwari, D., and Sandabe, M. K. 2010. ressource Naturelle. Wageningen: Centre for Agricultural

“Effect of Phosphorus Fertilizer on Nitrogen Fixation by Publishing and Documentation (Pudoc-DLO).

Some Grain Legume Varieties in Sudano—Sahelian Zone [20] Breman, H., and Niamngado, O. 1994. “Maintien de la

of North Eastern Nigeria.” Nigerian Journal of Basic and Production Agricole Sahélienne (Rapport Mi-chemin du

Applied Science 18 (1): 19-26.

Projet PSS).” Rapport PSS N. 6. [32] Pradhan, S., Chopra, U. K., Bandyopadhyay, K. K., Singh, [21] Bationo, A., and Ntare, B. R. 2000. “Rotation and

R., Jain, A. K., and Ishwar Chand 2013. “Effect of Water Nitrogen Fertilizer Effects on Pearl Millet, Cowpea and

and Nitrogen Management on Water Productivity and Groundnut Yield and Soil Chemical Properties in a Sandy

Nitrogen Use Efficiency of Wheat in A Semi-arid Soil in the Semi-arid Tropics, West Africa.” Journal of

Environment.” International Journal of Agriculture and

On-farm Evaluation of Effect of Organic and Mineral Fertilizers

592

on Biomass and Yield of Millet/Cowpea Intercrop in the Sahel, West Africa

Food Science Technology 4 (7): 727-732. [39] Yamoah, C. F., Bationo, A., Shapiro, B., and Koala, S. [33] Payne, W. A., Malcon, C. D., Hossner, L. R., Lascano, R.

2002. “Trend and Stability Analyses of Millet Yields J., Onken, A. B., and Wendt, C. W. 1992. “Soil

Treated with Fertilizer and Crop Residues in the Sahel.” Phosphorus Availability and Pearl Millet Water-use

Fields Crops Research 75: 53-62. Efficiency.” Crop Science 32: 1010-1015.

[40] Nwofia, G. E., Nwanebu, M., and Agbo, C. U. 2012. [34] Abdoulaye, T., and Lowenberg-DeBoer, J. 2000.

“Variability and Inter-relationships Between Yield and “Intensification of Sahelian Farming Systems: Evidence

Associated traits in Cowpea (Vigna unguiculata (L.) from Niger.” Agricultural systems 64: 67-81.

Walp) as Influenced by Plant Populations.” World [35] Perry, D. A., Amaranthus, M. P., Borchers, J. G., and

Journal of Agricultural Sciences 8 (4): 396-402. Brainerd, R. E. 1989. “Bootstrapping in Ecosystems.”

[41] Namniar, P. T. C., Rao, M. R., Reddy, M. S., Floyd, C. Bioscience 39 (4): 230-237.

N., Dart, P. J., and Willy, R. W. 1983. “Effect of [36] Manlay, R. J., Kaïré, M., Masse, D., Chotte, J. L., Ciornei,

Intercropping on Nodulation and N 2 -fixation by G., and Floret, C. 2002a. “Carbon, Nitrogen and

Groundnut.” Experimental Agriculture 19: 79-86. Phosphorus Allocation in Agro-ecosystems of a West

[42] Gueye, T., and Becker, H. 2011. “Genetic Variation in African Savanna, I. The Plant Component under

Nitrogen Efficiency among Cultivars of Irrigated Rice in Semi-permanent Cultivation.” Agriculture, Ecosystems

Senegal.” Journal of Agricultural Biotechnology and and Environment 88: 215-232.

Sustainable Development 3 (3): 35-43. [37] Manlay, R. J., Masse, D., Chotte, J. L., feller, C., Kaïré,

[43] Dobermann, A. 2006. “Nitrogen Use Efficiency in Cereal M., Fardoux, J., and Pontanier, R. 2002b. “Carbon,

Systems.” In Proceedings of the 13th ASA Conference. Nitrogen and Phosphorus Allocation in Agro-ecosystems

[44] Dugue, P. 1985. “Preparation du sol en Zone of a West African savanna, II. The Soil Component

Sahelo-soudanienne, Atouts et Contraintes.” In Under Semi-Permanent cultivation.” Agriculture,

FSUSAFGRAD sur les Technologies Appropriees Pour Ecosystems and Environment 88: 233-248.

les Paysans des Zones Semi-arides d’Afrique Occidentals. [38] Manlay, R. J., Chotte, J. L., Masse, D., Laurent, J. Y., and

Ouagadougou, Burkina Faso.

Feller, C. 2002c. “Carbon, Nitrogen and Phosphorus [45] Bationo, A., and Mokwunye, A. U. 1991. “Role of Allocation in Agro-ecosystems of a West African

Manures and Crop Residue in Alleviating Soil Fertility Savanna, III. Plant and Soil Components under

Constraints to Crop Production: With Special Reference Continuous Cultivation.” Agriculture, Ecosystems and

to the Sahelian and Sudanian Zones of West Africa.” Environment 88: 249-269.

Fertilizer Research 29: 117-125.

July 2014, Vol. 8, No. 7, pp. 593-602

Journal of Life Sciences, ISSN 1934-7391, USA

DAVID PUBLISHING

Detection and Protein Modelling for Consensus Region of hsp70 Gene Family in Egyptian Arundo donax

1 2 Mohamed A. Ezz 3 , Mona I. Salah and Abdullah I. Ammoura 1. GEBRI, Bioinformatics Department, Sadat University, P.O. Box 79/22857, Egypt

2. College of Biotechnology, Misr University for Scienceand Technology, 6th of October City, October 12451, Egypt 3. Molecular Genetics Lab, College of Biotechnology, Misr University for Scienceand Technology, 6th of October City, October

12451, Egypt

Received: April 18, 2014 / Accepted: July 14, 2014 / Published: July 31, 2014.

Abstract : Degenerate primers are particularly useful in amplifying homologous genes from different organisms. This paper describes a method for designing degenerate primers for a given multiple alignment of DNA sequences of hsp70 gene family using ClustalW algorithm and detect the consensus region in gene family of hsp70 in a plant species which have not any recorded information about hsp70 gene family in the Genbank of National Centre of Biotechnology Information (NCBI) like Arando donax .The sequenced consensus sequence of Arando donax is considered a gene marker in building the genome map sequence. The BLASTn program is used to find a homology between more than one accession numbers of DNA sequences, (X67711.2) was for Oryza sativa (hsp70), (AY372071.1) was for Nicotiana tabacum (hsp70) and (L41253.2) was for Lycopersicon esculentum (Hsc70). In silco PCR module was performed to detect the melting temperatures (Tm) and predicted the PCR product size (783 bp).The result of designed degenerate primers showed that there was a homology founded among the designed primers and the DNA templates of the recorded sequences (AY372071.1, X67711.2 and L41253.2) with at least 80% identity. The designed degenerate primers were used to isolate a consensus region of hsp70 gene family of Arando donax at the expected molecular weight (783 bp). The isolated PCR product, (783 bp) of Arando donax was sequenced and submitted to the Database of Japan (DDBJ) with accession number AB819871. The ORF finder tool translated the accession number AB819871 and gave a selected frame which used to build 3D structure model. In conclusion, this study focused on the importance of designing the degenerate primers to isolate the gene family and predict the 3D structure of gene family depending on the ORF finder tool of Genebank.

Key words : ClustalW tool, degenerate PCR, different melting temperatures, in silico PCR, SWISS-MODEL workspace.

1. Introduction  blocks of conserved regions in multiple global alignments. Therefore, alignment quality should be

The Hsp70s are found in all major cellular

very high [4].

compartments of eukaryotes and every bacterium Degenerate PCR is a PCR method that uses examined to date, bind partially unfolded proteins. degenerate primers to amplify unknown DNA They appear to bind nascent chains in the process of sequences that are related to a known DNA sequence protein synthesis and completed polypeptides upon or to amplify a mixture of related sequences in one release from ribosome [1]. Classical methods for PCR reaction. Degenerate primers are just a mix of degenerate primer design include software applications primers with similar sequences. Degenerate PCR is such as CODEHOP [2] or PrimaClade [3]. These useful for identify new members of a gene family or methods usually rely on the identification of clear orthologous genes from different organisms (http://

www.protocolonline.org/prot/Molecular_Biology/PCR/ Corresponding author: Mohamed A. Ezz, M.Sc., Ph.D. Candidates, research filed: molecular genetics. E-mail:

Degenerate_PCR___Degenerate_Primer). Degenerate ezz111@yahoo.com.

Detection and Protein Modelling for Consensus Region of hsp70

Gene Family in Egyptian Arundo donax

primers are particularly useful in amplifying (L41253.2), (AY372071.1), (X67711.2) and homologous genes from different organisms [5, 6]. In

(XM_003563568.1). The MegAlign module used the the “candidate gene approach”, known genes that

ClustalW algorithm to align more than one sequence affect similar processes in one organism could have

and produced the conserved blocks, which had been their homologues amplified in other related organisms

used to design the degeneracy primers. The setting by the use of a well-designed degenerate primer pair

parameters of ClustalW were adjusted (gap penalty [7]. The authors determined which parts are conserved

equal to 15, gap length penalty was equal to 6.66, and which are variable in the conserved blocks which

DNA transition weight equal to 0.6 and DNA weight produced by the ClustalW2 algorithm to design the

matrix was ClustalW). The definition of ClustalW degenerate primers to detect a consensus region of

parameters

hsp70 gene in Arando donax. The authors report a (1) Gap Penalty: the amount deducted from the new accession number for Arando donax,

alignment score for each gap in the alignment and AB819871.1 and identify all open reading frames for

Gaps of different sizes carry the same penalty. the accession number AB819871.1 by ORF finder tool

(2) Gap Length Penalty: the value deducted from and select the PROSITE pattern to retrieve an

the alignment score after first multiplying it by the alignment of UniProtKB/Swiss-Prot presented true

length of gaps. Longer gaps have a greater penalty positive hits with the selected frame. Authors predict

than shorter gaps.

the 3D structure of the selected frame. (3) For ClustalW only, you may specify values for Delay Divergent Seqs (%): alignment of any

2. Material and Methods

sequences with lower percent identities than this

2.1 Blasting by the Query Sequence threshold, with respect to other sequences in your The DNA sequence Oryza sativa hsp70 gene was

project, is delayed until after alignment of sequences retrieved from the sequence database of NCBI with

with higher percent identities.

gene information number gi|21664286 and accession (4) DNA Transition Weight: transitions are number (X67711.2), the FASTA file format was used

purine-purine (A-G) and pyrimidine-pyrimidine (C-T) as a query sequence to hit the database of Gene bank

substitutions. In DNA alignments, matches are usually and the category of Database that was chosen a (nt/nr)

scored 1, mismatches 0. The transition weight—allows nucleotide collection non redundant database and the

you to specify a score for transitions anywhere algorithm type of the BLAST (Basic Local Alignment

between 0 and 1. Increasing the transition weight Search Tool) was BLAST-nucleotide (BLASTn). The

towards 1 may help to make finer distinctions between three accession numbers which retrieved from closely related sequences. For distantly-related BLASTn and selected were (X67711.2) of Oryza

sequences, you may want to set this value to 0. sativa hsp70 gene, (AY372071.1) of Nicotiana

2.3 The Module of Primer Select

tabacum (HSP70-3) gene, (L41253.2) of Lycopersicon esculentum (Hsc70).

The module of primer select of DNASTAR lasergene program, version 7.0 was used to locate the

2.2 Detect the Conserved Block Sequence to Design specific primers. The location of consensus sequence the Degenerate Primers was assigned to be from the nucleotide position 2913

The MegAlign module of DNASTAR lasergene to 4557 (extracted conserved blocks from MegAlign version 7.0 was used to detect the consensus sequence

module) and the PCR product length was adjusted to between the previous four accessions numbers

be from 690 bp to 800 bp. All the parameter of primer

Detection and Protein Modelling for Consensus Region of hsp70

Gene Family in Egyptian Arundo donax

characteristics were adjusted from the primer locate tabacum (Samsun), Oryza sativa (Sakha102), menu of the program then the scoring option was

Lycopersicon esculentum (Castlerock) and leaves selected to adjust the values of set control to be in

samples of Arando donax were collected from intermediate including the following parameters,

Alexanderia, Egypt. The genomic DNA was isolated melting temperature (Tm), the primer length, hairpin

by the method CTAB (Cetyltrimethyl ammonium loop, overall stability and primer stability of 3’ end.

bromide according to the method [9]) for the three The default value of primer stability of 3’ end was

plants (Oryza, Tomato, Nicotina and Arando). The (-8.5 kCal/mol) is the average of all nearest neighbor

total volume of PCR reaction was equal to 25 µL (1.5 pentamer interactions, Primers with pentamer ΔG (the

µL of DNA with O. D 50-100 ng/µL), 0.5 µL for each amount of required energy to break the secondary

primer (Master Mix 12.5 µL of DreamTaq PCR structure in hairpin loop) more stable than -8.5

Master Mix (2X) and 10 µL free nucleases water). kCal/mol (more negative) have a tendency to false

The primers were synthesized at prime and are more likely to form hairpins and self

metabion-international. The PCR reactions were dimmers.

carried out using a DNA thermal cycler (Techne-312

2.4 Check for the Specificity and Uniqueness of the and Biometra-unoII) for every plant with different Degenerate Primers

melting temperatures Oryza sativa (94 o

C 2 min then

C 30 s, Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/

40 r as follows 94 o C 30 s, Tm 45 C to 47

C 10 min), Lycopersicon tools/primer-blast) [8], was used to detect the esculentum (94 o

72 o

C 1 min followed by 72 o

C 30 s, homology between the designed primers and targeted

C 2 min then 40 as follows 94 o

C 10 templates in the gene bank database and also the

Tm 45.3 o

C 30 s, 72 o

C 1 min followed by 72 o

C 2 min then 40 as position of degeneracy nucleotides and the uniqueness

min) and Arando donax (94 o

C 1 min for the forward and reverse primers. The parameters

follows 94 o C 30 s, Tm 45.0 C 30 s, 72

C 10 min). Amplified products were for Primer-BLAST were adjusted to be default. The

followed by 72 o

analyzed on a 2% agarose gel containing ethidium forward and reverse primers were putted through the

bromide. 100 bp DNA ladder have loaded, Biolabs. input text box of the Primer-BLAST homepage page

2.7 Sequencing the Expected Size of hsp70 Gene of (http://www.ncbi.nlm.nih.gov/tools/primer-blast).

Arando donax

2.5 PCR In silco Module to Predict and Produce All the True Condition of PCR Program

The PCR product was clarifid and sequencd using Genetic Analyzer (23 ABI 3730XLs) by the The PCR in silico module is a tool which

degenerate primers forward and reverse primers. The implemented the FAST PCR program by database search was performed using BLASTn

Ruslan-Kalendar version 4.0.8 university of Helsinki, program (http://blast.ncbi.nlm.nih.gov/BlastAlign. cgi). Finland and the PCR in silico module was used to

2.8 Detection of (ORF) by Using ORF Finder Tool for produce all the details of the PCR program like Tm

Accession Number AB819871.1

for the forward and reverse primer and the predict PCR product and informed the positions of primers

Analysis tool which finds all open reading frames which were aligned on the template sequences

of a selectable minimum size in a user’s sequence or (L41253.2, AY372071.1, X67711.2 and AB819871.1).

in a sequence already in the database. This tool

2.6 Genomic DNA Isolation and PCR Reaction identifies all open reading frames using the standard

or alternative genetic codes. The deduced amino acid The cultivars were grown in black soil Nicotiana

sequence can be saved in various formats and

Detection and Protein Modelling for Consensus Region of hsp70

Gene Family in Egyptian Arundo donax

searched against the sequence database using the

2.10 Retrieving PROSITE Documentations Analysis BLAST server (www.ncbi.nlm.gov/projects/gorf/).

and Producing Multiple Sequence Alignment The submitted accession number AB819871.1 of

The selected frame (frame -1, ORF from 72..602) Arundo donax was entered in the form of the ORF

sequence was used as query sequence to hit by the finder tool to find the best translated frame and run subjected family signatures of SCANPROSITE Database. BlASTp (Blast protien) to find the related protein Then selected PROSITE pattern to retrieve an domain family to construct the caldeogram depending alignment of UniProtKB/Swiss-Prot true positive hits on the BLASTp results. with the selected frame (frame -1, ORF from 72..602).

2.9 Submit Protein Sequences to Scan Them against the PROSITE Collection of Motifs

2.11 Predicting the 3D Structure of ORF Finder Results

PROSITE currently contains patterns and profiles The 3D structure of the selected frame (frame -1,

specific for more than a thousand protein families or ORF from 72..602) was predicted by using the server

domains. Each of these signatures comes with documentation providing background information on

Swiss-model (http://swissmodel.expasy.org/ interactive), which is a fully automated protein structure homology

the structure and function of these proteins (http://www.expasy.org/tools/scanprosite/scanprosite-

modelling server, accessible via the ExPASy web server, or from the program DeepView (Swiss

doc). PROSITE is a method of determining what is

Pdb-Viewer).

the function of uncharacterized proteins translated from genomic or cDNA sequences. It consists of a

3. Results and Discussion

database of biologically significant sites and patterns

3.1 Retrieving the Homologous Sequence by the formulated in such a way that with appropriate

BLAST Tool

computational tools it can rapidly and reliably identify which known family of protein (if any). The new

The BLAST program (Basic Local Alignment sequence belongs to (http://www.expasy.org/prosuser

Search Tool) produced a local alignment between the html#general). In some cases the sequence of an

query sequence (L41253.2) of Lycopersicon unknown protein is too distantly related to any

esculentum and the subjected records in the database protein of known structure to detect its resemblance

(AY372071.1) for Nicotiana tabacum, (X67711.2) for by overall sequence alignment, but it can be Oryza sativa. Each pairwise alignment (local identified by the occurrence in its sequence of a

alignment) had a bit score (normalized score for the particular cluster of residue types which is variously

score of the alignment) and the optimal score of known as a pattern, motif, signature, or fingerprint.

alignment (OPS). The distance of similarity between These motifs arise because of particular requirements

the query sequence and the targeted sequences had a on the structure of specific region(s) of a protein which

significance value or not, that depending on the may be important, for example, for their binding

E-value for every bit scores of pairwise alignment. properties or for their enzymatic activity The (E-value) reflected the significance of similarity (http://www.expasy.org/prosuser. html#general). The

between the query sequence and the targeted or accession number AB819871.1 of Arundo donax was

subjected sequences (Table 1). The homology results entered in the form of the link between the query sequence accession number (http://prosite.expasy.org/scanprosite) and the option

(L41253.2) of Lycopersicon esculentum and the

of submit protein sequences was selected to scan them subjected records in the database. The subjected

against the PROSITE collection of motifs. records which were selected (AY372071.1) for

Detection and Protein Modelling for Consensus Region of hsp70

Gene Family in Egyptian Arundo donax

Table 1 Sequences produce significant alignments.

Accession Description Max score E-value Max identity L41253.2

(query) Lycopersicon esculentum Hsc70 (Hsc70) gene, complete cds

0 100% X67711.2

0 80% AY372071.1 Nicotiana tabacum heat shock protein 70-3 (HSP70-3) gene, complete cds

Oryza sativa hsp70 gene for heat shock protein 70

and had some degeneracy nucleotides in the position from the position 2913 to 4557 that range was used to produce the degenerated primers pairs.

3.3 The Module of Primer Select The module of primer select of DNASTAR

Lasergene version 7 had analyzed the consensus sequence and the characteristics of primer design to produce a report of amplification summery which had obtained as shown in the (Fig. 1). The amplification

Fig. 1 The report of amplification summary.

summery described and documented the information about the designed primers, forward primer Nicotiana tabacum and (X67711.2) for Oryza sativa

GCTGCTGAAGAAATCTCCTCCAT and the reverse were homologous to query sequence and they were

primer AGCACCATAAGCAACAGCCTCAT where selected depending on their similarity of E-values

C and which were equal to zero for all the three accession

the (Tm) of forward primer was equal to 55.6 o

C. The numbers (L41253.2, AY372071.1 and X67711.2).

the (Tm) of reverse primer was equal to 56.8 o

primer Tm difference was equal to 1.2 o C.

3.4 Check for the Quality and Uniqueness of the Region

3.2 The Results of MegAlign Tool to Detect Conserved

Selected Primers and the Homology between the The homologous sequences were retrieved from the

Template Sequences and Primers

database we downloaded the FASTA format of the The quality and uniqueness of the forward and reverse three accession numbers (L41253.2, AY372071.1 and

primers were checked by using the Primer-BLAST X67711.2) in one text file and then the text file was

tool (http//www.ncbi.nlm.nih.gov/tools/primer-blast). processed by MegAlign tool of DNASTR Lasergene

The best alignment of forward and reverse primers version 7.0 to produce the multiple sequence alignment

with the three accession numbers (L41253.2, between the three accession numbers (L41253.2,

AY372071.1 and X67711.2). The Primer-BLAST tool AY372071.1 and X67711.2).Variant degrees of was used to check the uniqueness of the selected

conservation were found and the highly conserved primers and produced a summary table of melting blocks (Red Bar) was detected from the histogram bar

temperatures as shown in (Table 2). The analysis which indicated for the degree of the conservation in

number (3.4.1) showed the results of using colour metric scale. The red bar indicated for the best

Primer-BLAST2. The primer pair aligned and hit 180 positions of conserved sequences which were assigned

subjects, which recorded in the gene bank as hsp70 gene from the nucleotide position 3557 to 4491. The

or Hsc70 gene. The taxonomy of the recorded subjects, detected block was produced the consensus sequence

were distributed between monocots and dicots plants.

Detection and Protein Modelling for Consensus Region of hsp70

Gene Family in Egyptian Arundo donax

Table 2 Products on intended targets.

Sequence(5' →3') Length Tm GC% Forward primer

GCTGCTGAAGAAATCTCCTCCAT 24 61.48 41.67% Reverse primer

AGCACCATAAGCAACAGCCTCAT

3.4.1 The Results of Using Primer BLAST2

Template 1176 .........G.............. 1154

> X67711.2 Oryza sativa hsp70 gene for heat

3.5 Define the Degeneracy of the Nucleotide in the

shock

DNA Sequence

protein 70 product length = 783

The degenerate sequences in the aligned sequences

Forward primer 1

was obtained by the primer blast tool for the four

GCTGCTGAAGAAATCTCCTCCAT 23

accession numbers (X67711.2, L41253.2,

Template 2979 .....C.....G........... 3001

AY372071.1 and XM_003563568.1) and reported in

Reverse primer 1

the analysis number (3.4.1) then the mismatched bases

AGCACCATAAGCAACAGCCTCAT 23

between the primer and the templates were indicated

Template 3761 ....................... 373

for the degenerate bases and highlighted in gray

> L41253.2 Lycopersic esculentum (Hsc70) gene,

colour and reported in the analysis number (3.4.1).

complete cds

The colour of the degenerate bases were red

product length = 783

F-HSP70-AD KCWGCYGAAGARATYTCYTCYAT

Forward primer 1

R-HSP70-AD AGCWCCATARGCAACAGCCTCRT

GCTGCTGAAGAAATCTCCTCCAT 23

where K(G/T), W(T/A), R(A/G) and Y(T/C).

Template 2779 T...................T.. 2801

3.6 The Result of PCR In silico Module

Reverse primer 1 AGCACCATAAGCAACAGCCTCAT 23

The expected PCR product which had been

Template 3561 ...T.................G. 3539

predicted was equal to 783 bp for all the three

> AY372071.1 Nicotiana tabacum heat shock

accession numbers by the program Fast PCR version

protein 70-3 (HSP70-3) gene, complete cds

4.0. The PCR in silico module of Fast PCR program

product length = 783

was used to detect the true positions of the aligned

Forward primer 1

primers with the template sequences (L41253.2,

GCTGCTGAAGAAATCTCCTCCAT 23

AY372071.1, and X67711.2). Fast PCR program was Template 1419 .................T..... 1441 used to detect the true PCR Condition like Tm and the

Reverse primer 1

“%” of similarity between the primer pairs and the

AGCACCATAAGCAACAGCCTCAT 23

three accessions (L41253.2, AY372071.1 and

Template 2201 .........G............. 217

X67711.2). The “f” is for forward and the “r” is for

> XM_003563568.1 Brachypodium distachyon

reverse as shown in (Fig. 2a), (Fig. 2b) and (Fig. 2c).

heat shock cognate 70 kDa protein 1-like, transcript

3.7 Loading the PCR Products

product length = 783 Forward primer 1

The products of PCR for three plants (Tomato, Rice

GCTGCTGAAGAAATCTCCTCCAT 23

and Arando) had been detected approximately at M.W

Template 394 T.A...........T..T..... 416

size 783 bp as in (Fig. 3), DNA ladder from 100 bp to

Reverse primer 1

2000 bp had been loaded. Some bands are not the

AGCACCATAAGCAACAGCCTCAT 23

specific band that was referred to the designed low

Detection and Protein Modelling for Consensus Region of hsp70

Gene Family in Egyptian Arundo donax

result of ORF finder detection tool for the accession number (AB819871) achieved five frames. The selected frame (frame -1, ORF from (72..602)) gave a translated frame with Length: 176 aa which hit the subjected records in the Genbank and found distribution of 100 Blast hits (related to hsp70) on the query sequence and hit on the conserved domain

PTZ00009 [PTZ00009], heat shock 70 kDa protein

Fig. 2a PCR in silico result for Lycopersic esculentum—(L41253.2).

and achieved a significance E-value 2.44e-100.

3.9 Retrieving Prosite Documentations and Analyzing Family Signature 2 of Heat Shock hsp70 Proteins

The first technical section of the PROSITE documentation was selected, HSP70_2, PS00297 of heat shock hsp70 proteins family signature 2. The results were described as the PROSITE methods (with tools and information) and were covered in Swiss-Prot true The ClustalW format in condensed colour was

Fig. 2b PCR in silico result for Nicotiana

selected to see the degree of the conservation on the

tabacum—(AY372071.1).

motif HSP70_2, PS00329 of heat shock hsp70 proteins family. The retrieved results of HSP70_2 motif was [IFDLGGGTfdvSLL] as shown in the (Fig. 4a) and (Fig. 4b).

3.10 From Amino Acids Sequence to the 3D Structure The server was used to build an expected model

(http://swissmodel.expasy.org/interactive). The server was used to detect the high score of pair wise

alignment to compare the query sequence to the

curated 3D structure of the recorded protein in the

Fig. 2c PCR in silico result for Oryza sativa—(X67711.2).

protein data bank (PDB). Then calculated the

melting temperature from which were from 45 o

C to

secondary structure prediction of the amino-acids

47 o C Tm. sequence and finally to the right expected structure. The query sequence of amino acid (176 aa) was one of

3.8 Finding the Subjected Homologous Records to the the translated frame (frame -1, and ORF from

Accession Number (AB819871) (72..602)) of the accession number AB819871.1. The

The expected band of Arando donax was sequenced calculation of similarity among the query sequence of and submitted to the Database of Japan DDBJ and had

frame-1 (176 aa) of Arando donax and the targeted 3D the accession number (AB819871). The length of the

structures using the matrix of Blosum 62 and BLAST submitted nucleotide sequence was 764 and molecule

program were carried out to find the optimal score of type which was sequenced is genomic DNA. The

pair wise alignment, E-value and the overlap regions

Detection and Protein Modelling for Consensus Region of hsp70

Gene Family in Egyptian Arundo donax

Fig. 3 Amplified PCR of Genomic Products by using the designed degenerate Primer pairs to detect the gene of hsp70. Lane (2) product of Lycopersicon esculentum, Lane (3) product of Oryza sativa, Lane (4) and Lane (5) products of Arando donax. The expected sizes of PCR products at 783 bp as the red arrow assigned.

the score(GMQE) was 0.87 as shown in the (Fig. 5). GMQE (Global Model Quality Estimation) is a quality

estimation which combines properties from the

target-template alignment. The resulting GMQE score is expressed as a number between zero and one,

Fig. 4a The retrieved results of HSP70_2 motif was

reflecting the expected accuracy of a model built with

[IFDLGGGTfdvSLL].

that alignment and template. Higher numbers indicate

Fig. 4b The translated frame (-1) was scanned to detect the motif signal of HSP70_2, PS00329.

of pair wise alignment. The PDB number of 1kaz.1.A was one of the highest and the best score of pair wise

Fig. 5 The model_01 description and used the template

alignment between the PDB records and the query

1kaz.1 and query sequence achieved a high GMQE (0.87)

amino acid sequence (176 aa) and the significance of

for the expected template as the red circle indicated.

Detection and Protein Modelling for Consensus Region of hsp70

Gene Family in Egyptian Arundo donax

Fig 6 The alignment between the model_01 and the template 1Kaz.1.A and the ligand of (k+) ion in contact with chain A: D34, G36, T39, D41 as the red line indicated.

by a query sequence, the second step was used the BLASTn programme of NCBI (http://blast.ncbi.nlm. nih.gov/Blast.cgi) to find the homology between the query sequence and the subjected records which were homologous to the query sequence and also in the same gene family category, the third step was used the ClustalW algorithm to produce a multiple sequence alignment and the authors found the conserved block which had the reduced number of degenerate

nucleotides , the fourth step found and checked for the

Fig. 7 The model_01 was built with ProMod Version 3.70.

specificity of the degenerate primers which hit only higher reliability. Once a model is built, the GMQE

the subjected record of hsp70 gene family only and gets updated for this specific case by also taking into

finally the PCR in silco was performed and the account the QMEAN4 score of the obtained model in

selected melting temperature Tm was calculated and order to increase reliability of the quality estimation.

adjusted to be the annealing temperature which used and depending on the quality of GMQE the model

in the running of thermal cycler. The amino acids template—alignment was 1kaz.1.A which was sequence (176 aa) was one of the translated frame producing significant alignment and the expected

(frame -1) of the accession number, AB819871.1 and secondary structure of query sequence could be had

produced by using the ORF finder tool. The PDB ID the approximate secondary structure of the model

number, 1Kaz.1.A was homologous to the translated crystal structure 1kaz.1.A in PDB as shown in (Fig.

frame (frame -1) because the (GMQE) score of 6).The expected model_01 is a part of the chain A in

alignment between the expected model_01 and the template model 1Kaz.1.A as shown in the (Fig 7).

1Kaz.1.A template was a significance value and equal to 0.87. Depending on the score of Global Model

Quality Estimation (GMQE) the model_01 was the The authors report the use of such degenerate

4. Conclusion

expected secondary structure of the translated frame primers to detect the genes of hsp70 and from the

(frame -1). Model_01 was expected to be the same multiple DNA sequences, authors determined which

secondary structure of the crystallized 3D structure parts are conserved and which are variable. Based on

1Kaz.1.A.

this information, the conserved DNA motif used as

References

starting points to design degenerate PCR primers and [1] Graig, E. A., Gambil, B. D., and Nelson, R. J. 1993.

the main steps were concluded in six steps of using “Heat Shock Proteins Molecular Chaperones of Protein bioinformatics tools began with hitting the database

Biogenesis.” Microbiol Rev. 57 (2): 402-414.

602

Detection and Protein Modelling for Consensus Region of hsp70

Gene Family in Egyptian Arundo donax

[2] Rose, T. M., Henikoff, J. G., and Henikoff S. 2003. Arabidopsis Thaliana.” Mol. Plant Microbe Interact 11 “CODEHOP (COnsensus-DEgenerate hybrid

(4): 251-258.

oligonucleotide primer) PCR Primer Design.” Nucleic [6] Shen, K. A., Meyers, B. C., Islam-Faridi, M. N., Chin, D. Acids Research 31 (13): 3763-3766.

B., Stelly, D. M., and Michelmore, R. W. 1998. [3] Gadberry, M. D., et al. 2005. “Primaclade—a Flexible

“Resistance Gene Candidates Identified by PCR with Tool to Find Conserved PCR Primers Across Multiple

Degenerate Oligonucleotide Primers map to Clusters of species.” Bioinformatics 21 (7): 1263-1264.

Resistance Genes in Lettuce.” Mol. Plant Microbe [4] Gorrón, E., et al. 2010. “A New Method for Designing

Interact 11: 815-823.

Degenerate Primers and Its Use in the Identification of [7] Deng, C. a. T. M. D. 2001. “Molecular Identification of Sequences in Brachiaria Showing Similarity to

the Yellow Fruit Color (c) Locus in Diploid Strawberry.” Apomixis-Associated Genes.” Bioinformatics 26 (16):

Theoretical and Applied Genetics 103: 316-322. 2053-2054.

[8] Rozen, S., and Skaletsky, H. 2000. “Primer3 on the [5] Aarts, M. G., Lintel Hekkert, B. te, Holub, E. B., Beynon,

WWW for General Users and for Biologist J. L., Stiekema, W. J., and Pereira A., et al. 1998.

Programmers.” Methods Mol. Biol. 132: 365-386. “Identification of R-gene Homologous DNA Fragments

[9] DOYLE, J. J. a. D. 1990. “J.L Isolation of Plant DNA Genetically Linked to Disease Resistance Loci in

from Fresh Tissue.” Focus 12: 13-15.

July 2014, Vol. 8, No. 7, pp. 603-604

Journal of Life Sciences, ISSN 1934-7391, USA

DAVID PUBLISHING

The Emergence of Hybrid Seeds and Polyembryony in Some Citrus Cultigens

Nino Davit Kipiani Department of Agricultural Sciences, Kutaisi State University, Kutaisi, 4600, Georgia

Received: April 18, 2014 / Accepted: July 14, 2014 / Published: July 31, 2014.

Abstract: An experiment was conducted and through distant hybridization a new promising forms of citruses were obtained. Namely, after hybridization of lemon Georgian and broad-leaved mandarin unshiu (mother components) with early-ripening trifoliata and a hybrid of citrus ichangensis—“Caucasus” (father components) a wide range of nucellar seedlings were obtained. It should be noted that nucellar seedlings of hybrid nature are practically closer to distant hybrids of sexual origin that are sharply inclined to the side of the mother component and produce good quality fruit. Unfortunately, such hybrids are either very rare or cannot be obtained at all. While, in the case of the study, through using new combinations in hybridization, the possibility of getting nucellar seedlings of the hybrid nature is much bigger. Distant hybridization turned out to be particularly interesting because of the polyembryony of citrus plants. Due to this, it gives opportunity to get new promising forms not only from the egg cell but from nucellar cells as well.

Key words: Distant hybridization, polyembryony, nucellar and hybrid seedlings.

1. Introduction  through distant hybridization.

In order to get nucellar seedlings of hybrid nature, Citruses are the best objects for studying the events the flowers of lemon Georgian and broad-leaved of polyembryony. As it is known that citruses and mandarin unshiu were pollinated with the pollen from other citrus plants are characterised by the trifoliata (early-ripening) and a hybrid of citrus polyembryony, i.e., when in one ovule two or more ichangensis—“Caucasus”. Hybrid seeds were sown embryos are developed. Consequently, from one seed and the obtained hybrid seedlings were genetically two or more plants appear that is connected to the studied from the moment of the emergence of hybrid development of additional embryo in nucellar cells. seedlings to the end of two vegetation periods. [2]. It Embryos developed from nucellar cells are called should be noted that it is possible to distinguish the nucellar embryos and the plants grown from such seedlings of sexual hybrids from nucellar ones by embryos are referred as plants of nucellar origin or external phenotypic signs: leaf shape, size and its nucellar seedlings [1]. down, the smell of a leaf, etc. These signs are

2. Materials and Methods

phenotypic but they are genotypic (inherited) as well. These were the characteristic features used when

The experiment was conducted in Laboratory of comparing the initial seedlings with their parents. The Citrus Genetics and Selection within the framework of seedlings which have the above mentioned male-parent the national scientific grant project. The aim of the phenotypic characteristics were considered to be project was to obtain new promising forms of citruses sexual hybrids and were grouped separately while the

rest (similar to a mother) were referred as nucellar Corresponding author: Nino Davit Kipiani, Ph.D., assistant

professor, research fields: Agriculture, Potential Possibilities of seedlings and were grouped separately. The results of Distant Hybridization in Citruses. E-mail: the study are given in the Table 1 below:

nino.kipiani74@yahoo.com.

The Emergence of Hybrid Seeds and Polyembryony in Some Citrus Cultigens

Table 1 The Emergence of hybrid seeds and polyembryony.

The number of emerged

Including

The number of

# Combination

seedlings

sown seeds

Hybrid seedlings

Nucellar seedlings

pieces % 1. Broad-leaved Mandarin Unshiu × Trifoliata (early-ripening) 100

pieces

pieces %

62 48 77.4 14 22.6 Broad-leaved Mandarin Unshiu

2. × Citrus

73 58 79.5 15 19.5 ichangensis—“Caucasus” 3. Georgian Lemon × Trifoliata (early-ripening)

90 68 75.6 22 24.4 4. Georgian Lemon × Citrus ichangensis—“Caucasus”

The data given in the table clearly show that the seedlings is higher than in case of other combinations. possibility of obtaining nucellar seedlings in citruses

The percentage difference between combinations to through the distant hybridization is generally low and

obtain nucellar seedlings is not very big but it's it varies according to the hybridization combinations.

necessary to constate it since the possibilities of The analysis of the material enabled to detect a

obtaining nucellar seedlings are limited and apparently, common pattern. Although there is no big difference

are conditioned by the biological characteristics between them, but the ability of seeds to emerge from

specific to species.

hybrid seeds in both combinations of Citrus