Foreword

Potato tubers as a valuable food product for most people in the world are considered “second bread”.

   Agricultural environment of mountainous areas of Tajikistan situated at an altitude of more than 1800 meters above sea level make it possible to grow good and quality harvest of potato.  Under these cool mountainous conditions potato plants are of little affection, have intensive bloom, and form a lot of berries and botanic seeds.

           During the researches made in different soil and climatic conditions a number of scientists ( R.L.Perlova  (1958), N.N.Balashev  (1968), N.V.Lebedeva  (1970), S.M.Bukasov , A.Ya.Kameraz  (1972), I.M.Yashina, I.P.Sklyarova, V.P.Kiryukhin (1983), Jongirov J.О (1995), H.A.Mumindjanov (2003), Shpaar D. (2004), S.D.Kiru  (2007), K.Partoev,  B.Karimov,  M.Sulangov, K.Melikov  (2007), Carli C., D. Khalikov, F.Yuldashev, K. Partoev, K. Melikov, S. Naimov (2008) and K.Partoev, M. Sulangov, K. Melikov, S. Naimov, K. Aliev, Z. Davlatnazarova, B. Karimov, T. Mukimov (2008)) have ascertained the significance of breeding pair selection and locality of crossbreeding so as to get good results of potato hybridization. 

According to the following breeders: A.O. Mendiburu, S.J.Peloquin (1976); Frankel,R and E. Galun, (1977), SK. Kaushik, R.K. Birhman, B.P.Singh, J.Gopal (1997), Gopal,J. (1994), R.Kumar and J.Gopal (2003), S.K. Pandey, S.V.Singh, S.K.Chakrabarti, P.Manivel (2003) V.K Gupta, K.C. Thakur, Shantanu Kumar, S.K.Pandey, Uma Sah (2004) S.K. Luthra, S.K. Pandey, B.P.Singh, G.S.Kang, S.V. Singh and P.C. Pandey (2006), Partoev K., Naimov S., Melikov K., Jumahmadov A., Abdurahimov A. (2009) success of selective and genetic work aimed at nurture of new perspective grades depends to a large extent on pollen grain fertility and vitality when conducting different crossing between grades and species of potato.

Thus, on basis of above researchers work analysis and our experience on potato crossbreeding obtained in India this year, for the first time in the highland of Tajikistan we have begun to research the rate of pollen grain fertility and to cross different grades and hybrids of potato and the results of this researchers brining in this brochure. 

 2.1. Methodology and subjects of inquiry

To study the methods and ways of identification of pollen grain fertility and conducting potato crossbreeding, during the first two weeks of July, 2009, with the assistance of the International Potato Centre (CIP) we succeeded in visiting the Central Potato Research Institute (CPRI) in Shimli, India, (K.Partoev).  During this visit, such experienced Indian selectionists like Dr. Pandey S.Kumar, Jay Gopal, Vinod Kumar, Niradj Sharma, Lutra Kumar, Shambu Kumar, S.Chakrabari and others accessibly trained us in methods of estimating pollen grain vitality and conducting potato cross.  The above methods have been used in our research work in Tajikistan.

To identify vitality of potato pollen grains the following types of work should be prepared and carried out:

• To make acetocarmine solution, dropping bottle, forceps and needle ready for potato pollen release.
• To remove flower pistil using forceps.
• To pour out the pollen on the slide in the acetocarmine drop by shaking the stamen filament of the flower.
• To cover the acetocarmine and pollen mixture with a glass sheet.
• The ready preparation is to be examined through the microscope in 3-5 minutes.
• The quantity of acetocarmine coloured pollen grains (both fertile – vital pollen grains and yellow pollen grains – sterile or unviable grains) should be counted in three or five microscopic fields.
• The counted quantity of coloured and uncoloured pollen grains is to be recorded into the log book.

In some cases when shaking the stamen filament pollen grains are not poured out. It is observed when the blooms or anthers are not mature enough or can be also connected with genetic character of potato grades and species. In that case one or two anthers are taken and by means of forceps and needle pollen grains are released and mixed with an acetocarmine drop on the slide, following which anthers remains are removed and the preparation is getting ready to be examined on the glass sheet cover.

Pollen vitality was analyzed in three terms: on July, 20 (the beginning of blooming period), on August, 1 (mass blooming period) and on August, 10 (the end of blooming period) in 2009. In each term the pollen of five plants of each sample was taken, 15 plants in total. Fertile (vital) and sterile (unviable) pollen grains were counted in three microscope fields with increasing of 8x7 on the microscopes MBC-9 and MBC-10 and with increasing of 20x7 on the microscope MBU- 4А.

      As a material for research 62 clones and varieties of potato that were kindly given by the International Centre of Potato Investigation (CIP & CGIAR) in the network of plant gene pool investigation in the republics of Central Asia and the Caucasus were served. Also the collection of potato samples obtained by the  Institute of pant physiology and genetics of the Academy of Sciences of the Republic of Tajikistan and Social Organization “Tukhmiparvar” from VIR in 90-th years of the last century. Sample planting was made on March, 13, 2009 at an altitude of 2700 meters above sea level on the field station of Social Organization “Tukhmiparvar” situated in Jirgital region, Sari-Kendja village, Muksu jamoat, republic of Tajikistan.

      Here we also carried out straight and back crossing of different potato grades and species for obtaining first generation hybrids. For conducting potato grades crossbreeding after 3 p.m. emasculation of maternal plant flowers (removal of flower stamen filament with forceps) was made. After 4 p.m. we plucked paternal plant flowers, removed their pistils and kept them in the room temperature environment from 4 p.m. to 9 a.m. of the next day in Petri dish.

       In the morning, inside the room we shook stamen filaments with anthers using forceps and needle and picked the pollen into the glass Petri dishes of small size. The picked pollen was used for pollination of emasculated flowers by touching the pistil head with the picked pollen of paternal flowers. After pollination we attached a paper label indicating hybrid combination (maternal form x paternal form) and the date of crossbreeding written in pencil to the stem. The number of pollinated flowers was recorded into the potato crossbreeding log book in compliance with the dates of pollination (? Кардинал х ? Зарина– 25.VII. 2009).

 

2.2.  Findings of investigation

Investigations concerning identification of potato pollen grain fertility have been conducted in Djirgital region at an altitude of 2700 meters above sea level where an experimental plot of the Social Organization “Tukhmiparvar” and the Institute of Plant Physiology and Phytogenetics of the Academy of Sciences of the Republic of Tajikistan is situated. In the course of the investigations we managed to ascertain that fertile (vital) potato pollen grains placed in the acetocarmine drop are coloured and turned red. As to sterile pollen grains, they are not colored and will have yellow colouring. Their number can be easily counted through the microscope (Fig.1).

 

 Thus, by means of the above methodology for the first time in our republic we have estimated pollen grain fertility of potato grades and species. According to our researches pollen grain fertility of potato clones and grades in highland environment of our republic is genotypic ally specific. Most of the examined clones and grades have more than 80-97 per cent of fertile (vital) pollen grains. At the same time it is necessary to note that pollen grain fertility of some potato grades makes only 5-10 per cent (Table 2.2.1).

Table 2.2.1. Rate of fertile pollen grains of potato grades in highland environment (Jirgital region at an altitude of 2700 meters above sea level)

№	Varieties of potato	Analysis implementation dates:			Mean
		Jul. 20	Aug.1	10
1	Cardinal	28.0	24.4	27.1	26.5
2	Zarina	79.2	83.0	84.0	82.1
3	Dusti	95.0	96.6	94.0	95.2
4	Faizabad	85.9	86.0	84.1	85.3
5	Zhukovski	85.7	93.3	90.3	89.8
6	Condor	83.0	86.3	85.0	84.8
7	Piсasso	86.4	85.2	84.1	85.2
8	Hybrid 22	84.5	87.5	90.4	87.5
9	Hybrid 23	64.7	74.9	98.2	79.3
10	Clone 27/5	64.7	63.5	63.6	63.9
11	Clone  30/9	76.7	77.0	78.9	77.5
12	Clone  36/6	66.7	68.3	70.3	68.4
13	Clone  37/2	84.1	86.2	93.8	88.0
14	Clone  40/1	79.3	81.0	87.3	82.5
15	Clone  47/4	93.4	91.5	85.9	90.3
16	Clone  47/8	83.3	85.1	89.8	86.1
17	Clone  47/11	91.7	90.2	89.7	90.5
18	Clone  50/7	85.9	85.5	86.7	86.0
19	Clone   50/9	87.4	87.6	84.1	86.4
20	Clone  52/6	75.8	73.6	74.6	74.7
21	Clone  1	94.4	88.4	97.5	93.4
22	Clone  2	5.6	7.3	6.7	6.5
23	Clone  3	5.3	6.0	8.0	6.4
24	Clone  4	62.0	63.0	60.5	61.8
25	Clone  7	5.7	5.2	6.3	5.7
26	Clone  8	95.0	97.9	98.0	97.0
27	Clone 11	11.5	12.0	11.3	11.6
28	Clone 13	95.7	97.5	90.0	94.4
29	Clone 14	10.9	11.6	8.0	10.2
30	Clone 15	5.6	7.2	7.3	6.7
31	Clone 18	81.0	80.5	75.7	79.1
32	Clone 21	87.2	94.7	82.7	88.2
33	Clone 22	85.8	84.9	91.5	87.4
34	Clone 24	47.5	38.8	42.6	43.0
35	Clone 25	94.5	98.0	90.9	94.5
36	Clone 26	75.4	73.1	75.1	74.5
37	Clone 27	90.0	88.6	92.1	90.2
38	Clone 40	32.0	35.9	33.8	33.9
39	Clone 42	73.0	70.8	72.9	72.2
40	Clone 43	83.5	84.8	75.6	81.3
41	Clone 45	96.8	88.9	86.2	90.6
42	Clone 48	5.7	4.8	5.6	5.4
43	Clone 50	9.2	10.0	9.0	9.4
44	Clone 53	95.7	97.1	84.2	92.3
45	Clone 54	89.3	87.8	94.0	90.4
46	Clone 55	90.7	89.9	90.5	90.4
47	Clone 56	95.2	97.5	96.8	96.5
48	Clone 58	10.6	9.1	9.3	9.7
49	Clone 59	88.4	86.0	93.0	89.1
50	Clone 60	87.6	89.4	88.1	88.4
51	Clone 63	88.8	82.8	92.1	87.9
52	Clone 64	5.0	6.6	7.0	6.2
53	Clone 65	5.2	4.7	5.4	5.1
54	Clone 66	96.6	95.0	94.0	95.2
55	Clone 67	6.0	7.1	7.1	6.7
56	Clone 68	23.6	25.0	25.0	24.5
57	Clone 71	23.9	21.0	19.8	21.6
58	Clone 73	78.0	76.0	76.5	76.8
59	Clone 75	95.4	94.9	91.9	94.1
60	Clone 76	97.9	85.6	81.1	88.2
61	Clone 79	95.3	89.0	85.4	89.9
62	Clone 80	13.0	11.8	13.0	12.6
X		64.9	64.7	64.9	64.8
S		34.3	34.2	34.3	34.2
Sx		4.3	4.2	4.2	4.2
V,%		52.9	52.8	52.6	52.5
НСР 05		8.6	8.4	8.6	8.5

 

Among potato varieties cultivated in the mountainous area Сardinal grade had the least per cent of fertile pollen grains – 26, 5%. The new perspective Dusti grade had the greatest per cent of those – 95, 2%. This grade was raised by the scientists of the Institute of Plant Physiology and Phytogenetics of the Academy of Sciences of the Republic of Tajikistan, the Social Organization “Tukhmiparvar” and the International Potato Centre (CIP). This year the grade has been given to the Tajik State Commission on quality testing and grade protection.

      It should be noted that some clones sampled individually from hybrid swarm F₁ and propagated from one plant by culture method (in-vitro plants) had stamen filaments of different shapes. For example, clones 43, 48 and 54 had different morphological attributes of stamen filaments (Fig.2). Clone 43 had plants with two types of stamen filament shape. One part of the plant had a regular stamen filament; the other part of the plant had a modified stamen filament when stamen filaments are not pressed to the pistil stem as it is with regular plants. They are detached from it and look friable.

 

            Thus, according to the stamen filament shape among these new potato clones we can observe definite variation. It should be noted that the rest morphological signs (color of flowers and plants, leaf shape, evolution phenological stage and tuber color of the examined clones) remained stable. In addition, the researches indicated that the plants with modified shape of the stamen filament have weak yellow color of the stamen filament and low fertility of pollen grains compared to regular plants (Table 2.2.2.).

Table 2.2.2. Pollen grain fertility subject to the shape of the flower stamen filament, %

№	Varieties of potato	Analysis implementation dates:			Mean
		Jul. 20	Aug.1	10
1	Clone 43 (plants with regular stamen filament)	83.5	84.8	75.6	81.3
2	Clone 43 (plants with  modified shape of stamen filament)	45.7	50.1	47.5	47.8
3	Clone 48 (plants with  modified shape of stamen filament)	5.7	4.8	5.6	5.4

 

As you can see from above data (Table 2.), plants with modified shape of stamen filament have considerably less fertile pollen grains compared to those with regular stamen. The sign of cytoplasm male sterility is greatly detected with clone 48 where pollen grain fertility was only 5,4%. Along with it we have ascertained that although many potato clones have regular stamen filament shape and bright yellow coloring of the stamen filament and anther their index of fertility is very low. They are clones 2, 3, 7, 15, 50, 58, 64, 65 and 67 that have only 5,1 – 9,7 per cent of fertile pollen grains.  These clones can serve a good base line for potato breeding through straight crossing (without anther removal) and obtaining botanic seeds without flower emasculation. Thus, specific potato clones with male sterility sign have been defined.

As a result of potato hybridization carried out in Jirgital region from July 19 to August 11 (1650 crossings) by early September we have got six kg regular berries F₁, which seeds will be studied in 2010.                                                                                        

Table 2.2.3. The number of pollinated flowers and set berries obtained through potato hybridization (2009)

                                                                                                               

№	Maternal form	Paternal form	Number of pollinated flowers		Date of crossing		Number of gathered berries		Rate  of berry formation
1	Clone 75	Kufri Sadabakhar	18		19		0		0
2	Clone 76	Kufri Jirgari	48		19		18		37.5
3	Clone 66	Kufri Jiota	30		19		10		33.3
4	Khahadin	Kufri Arunda	38		19		3		7.9
5	Clone 75	Kufri Anchan	15		19		0		0
6	Clone 75	Kufri Suraya	34		19		6		17.6
7	Clone 59	GTH/107	26		19		0		0
8	Clone 66	Kufri Suraya	7		19		2		28.6
9	Clone 76	Mix of pollen	29		19		5		17.2
10	Clone 68	Mix of pollen	14		19		0		0
11	Clone 63	Mix of pollen	12		19		3		13.6
12	Clone 75	Clone 76	25		19		6		24.0
13	Clone 76	Clone 75	22		19		8		36.4
14	Clone 79	Clone 66	7		19		0		0
15	Zhukovski ranniy	Dusti	72		Jul. 21		0		0
16	Dusti	Zhukovski raniy	15		Jul. 21		0		0
17	Cardinal	Dusti	70	Jul. 21		3		43.0
18	Dusti	Cardinal	20		Jul. 21		0		0
19	Dusti	Condor	191		Jul. 21		83		43.5
20	Zarina	Dusti	40		Jul. 21		0		0
21	Dusti	Picasso	44		Jul. 21		17		38.6
22	Hybrid 23	Dustї	23		Jul. 21		0		0
23	Clone 40/1	Dusti	22		Jul. 21		1		4.5
24	Clone 40/1	Cardinal	17		Jul. 22		0		0
25	Hybrid 22	Dusti	11		Jul. 23		0		0
26	Hybrid 23	Picasso	53		Jul. 23		0		0
27	Faizabad	Picasso	127		Jul. 23		41		32.3
28	Cardinal	Condor	125		Jul. 31		88		70.4
29	Cardinal	Picasso	105		Jul. 31		84		80.0
30	Clone 48	Dusti	65		Jul. 31		2		3.1
31	Clone 40/1	Dusti	22		Jul. 31		16		72.7
32	Cardinal	Dusti	50		Aug. 1		0		0
33	Hybrid 23	Dusti	85		Aug. 1		0		0
34	Clone 40/1	Dusti	30		Aug. 1		6		20.0
35	Zarina	Hybrid 23	25		Aug. 1		2		8.0
36	Dusti	Condor	85		Aug. 11		53		63.4
37	Clone 48	Condor	28		Aug. 11		14		50.0
	Total:		1650				471		28.5

 

Summary:

For the first time in the mountainous area of Tajikistan study of pollen grain fertility and hybridization of potato varieties (Solanum tuberosum L.) have been carried out. The degree of pollen grain fertility and great variability of this genetic sign among 62 potato samples (5-97%) has been identified. It has been stated that some potato clones have great variability according to flower and anther coloring and flower stamen filament shape. Ten potato clones with male sterility sign and less quantity of fertile pollen grains (5,1 – 9,7%) have been picked out. Such clones can be a good material for selection and genetic work aimed at raising of new potato hybrids and grades. Among cultivated potato varieties Сardinal grade has the least per cent of fertile pollen grains – 26, 5% and the new perspective Dusti grade has the greatest per cent of those – 95, 2%. As a result of potato varieties and hybrids crossings 6 kg of hybrid berries have been obtained which seed will be sown and studied in 2010.