ISSN 00124966, Doklady Biological Sciences, 2014, Vol. 454, pp. 59–61. © Pleiades Publishing, Ltd., 2014. Original Russian Text © K.K. Sidorova, V.K. Shumnyi, 2014, published in Doklady Akademii Nauk, 2014, Vol. 454, No. 5, pp. 612–614.
GENERAL BIOLOGY
Symbiotic Mutants of Pea (Pisum sativum L.), an Important Genetic Source for Selection for Increased Nitrogen Fixation K. K. Sidorova and Academician V. K. Shumnyi Received October 31, 2013
DOI: 10.1134/S0012496614010190
hypernodulation genes (figure) [1–4]. Supernodulat ing mutant k301 marked by the gene nod4 was charac terized by abundant nodulation. It formed small nod ules all over the root throughout the vegetation period and demonstrated a high efficiency of nitrogen fixa tion. The mutant was characterized by a low seed yield compared to the initial cultivar Ramonsky 77. Super nodulating mutant k12 marked by the gene nod3 was induced from the cultivar Rondo. It also exhibited a high efficiency of nitrogen fixation and low productiv ity in comparison with the initial cultivar. The differ ence between these supernodulating mutants is that the mutant induced from the cultivar Ramonsky 77 had a fasciated stem, while the mutant obtained on the basis of the cultivar Rondo had a compact stem. The
An increase in the efficiency of the legume–rhizo bium symbiosis as an ecologically friendly and eco nomically inexpensive source of nitrogen taken up from the atmosphere is believed to be a challenging task of modern biology. In the symbiotic system, the macrosymbiont plays the role of an energy source, spending the products of photosynthesis not only on the production process (yield), but also on the forma tion of root nodules and nitrogen fixation. A microsymbiont consists of nodule bacteria that contain the enzyme nitrogenase, which reduces molecular nitrogen from the atmosphere to form ammonium. To increase the efficiency of nitrogen fix ation, selection may be carried out either at the level of the macrosymbiont via the development of legume genotypes characterized by a high productivity, intense nodulation, and nitrogen fixation or at the level of microsymbionts, the selection of which is aimed at the development of effective strains of nodule bacteria.
1
2
The goals of the present study were (1) the obtain ing and genetic study of a pea plant forms with improved symbiotic traits, such as active nodulation and nitrogen fixation and (2) the development of methods to use these forms for selection. The scientific novelty of this study is that we were the first to develop a method of the use of symbiotic mutants of pea plants for the selection aimed at the intensification of nitrogen fixation. The method is based on the combination of the alleles of different sym genes (the dominant hypernodulation gene Nod5 and the recessive supernodulation gene nod4) in the same genotype. Using the method of experimental mutagenesis, we obtained symbiotic mutants of pea plants, which were further used for identification of supernodulation and
Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, 630090 Russia
The root system of symbiotic mutant pea plants: 1, super nodulation; 2, hypernodulation.
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SIDOROVA, SHUMNYI
Table 1. Productivity parameters (M ± m) of local endemic forms of pea plants from Syria and Egypt and the recurrent lines of the F5 generation obtained as a result of the following crossbreeding: , a recurrent line of the F7 generation marked with the supernodulation gene nod4, which was ob tained by the crossing of the cultivar Druzhnaya with the su pernodulating mutant k301 (nod4) × , a local endemic form Per plant Variant
number of seeds
weight of seeds, g
192.2 ± 3.1
36.5 ± 7.1
6.7 ± 0.8
218.7 ± 12.9 214.1 ± 10.4 254.6 ± 5.3* 194.8 ± 9.0 160.7 ± 4.5
65.7 ± 13.1* 40.5 ± 5.8 113.0 ± 12.1* 48.2 ± 5.8 43.8 ± 4.6
14.1 ± 2.9* 9.7 ± 1.5 16.8 ± 1.6* 8.0 ± 1.1 4.5 ± 0.3
height, cm Local endemic form from Egypt Recurrent lines: ** k6ed k7ed k12ed k26ed Local endemic form from Syria Recurrent lines: ** k2sd k11sd
205.6 ± 14.6* 89.1 ± 14.9* 13.2 ± 1.7* 190.0 ± 5.5* 54.2 ± 3.8 7.5 ± 0.5*
* Difference from the parental endemic form is significant at p < 0.05; ** the number of recurrent lines is given in accordance with the Catalogue provided by the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences.
mutant carrying the dominant gene Nod5 was obtained on the basis of the cultivar Rondo. It is char acterized by hypernodulation and large nodules, which are located mostly at the upper part of the root. The mutant exhibited a high efficiency of nitrogen fix ation. The seed productivity of this mutant was higher
than that of the initial cultivar Rondo. We first identi fied the dominant gene Nod5 in the ancient pea plant cultivar Torsdag [4]. Currently, intense studies aimed at the develop ment of methods to use the symbiotic mutants for selection of pea plants in order to increase the effi ciency of nitrogen fixation are carried out. The use of the supernodulating mutant k301 (nod4), which was obtained on the basis of cultivars Ramonsky 77 and Torsdag and was marked by the gene Nod5, allowed us to develop the method of recurrent selection aimed at increasing in the efficiency of nitro gen fixation and high productivity. The method is based on the combining of different alleles of different genes within one genotype. The effectiveness of this approach was confirmed in experiments with feeder pea plant cultivars Druzhnaya and Novosibirskaya 1 [5]. Crossbreeding of local endemic forms of pea plants that originated from Syria and Egypt with the supern odulating mutant k301 (nod4) resulted in high levels of nodulation and nitrogen fixation in all recurrent lines of the F5 generation. However, all lines had a low seed yield. A positive result was achieved when the recur rent line of the F7 generation marked with the same supernodulation gene nod4 was used for crossbreeding instead of the mutant k301 (nod4). The line was obtained by crossbreeding of the feeder pea plant cul tivar Druzhnaya with the supernodulating mutant k301 (nod4) (Table 1). The results of the experiment showed that geno typic environment affected the expression of the supernodulation gene. Crossbreeding of two symbiotic mutants of pea plants, the hypernodulating mutant k74 (Nod5) and supernodulating mutant k12 (nod3), which were obtained on the basis of one cultivar Rondo, resulted in segregation of the F2 generation into supernodulat
Table 2. Symbiotic traits and productivity of supernodulating and hypernodulating mutants of pea plants (M ± m)
Variant
Symbiotic traits
Productivity
per plant
per plant
number of nodules
nitrogenase activity, nmole C2H4/h
height, cm
number of beans
number of seeds
Cultivar Rondo
138 ± 12
1269 ± 234
47.4 ± 0.7
6.6 ± 0.3
33.9 ± 1.8
Supernodulating mutant k12 (nod3)
802 ± 44
3166 ± 316
39.7 ± 0.8
5.4 ± 0.4
25.3 ± 1.5
Hypernodulating mutant k74 (Nod5)
380 ± 44
3425 ± 515
56.7 ± 2.1
11.5 ± 1.6
47.4 ± 7.2
Hypernodulating line obtained in re sult of the crossbreeding k74 × k12
1932 ± 29
8521 ± 480
69.4 ± 0.8
13.6 ± 1.6
67.4 ± 9.0
The results of the study show that both hyper and supernodulating mutants of pea plant can be used for selection aimed at the intensification of nitrogen fixation as a source for accumulation of nitrogen in soil. DOKLADY BIOLOGICAL SCIENCES
Vol. 454
2014
SYMBIOTIC MUTANTS OF PEA
ing and hypernodulating plants. The latter were char acterized by effective nodulation, active nitrogen fixa tion, and high productivity (Table 2). The results of our study demonstrate that symbiotic hyper and supernodulating mutant pea plants can be used in selection for an enhanced nitrogen fixation as a source of soil nitrogen accumulation. REFERENCES 1. Sidorova, K.K. and Uzhintseva, L.P., Dokl. Akad. Nauk, 1994, vol. 336, no. 6, pp. 847–849.
DOKLADY BIOLOGICAL SCIENCES
Vol. 454
2014
61
2. Sidorova, K.K. and Shumnyi, V.K., Dokl. Biol. Sci., 1997, vol. 353, no. 5, pp. 190–191. 3. Sidorova, K.K., Shumnyi, V.K., and Mishchen ko, T.M., Dokl. Biol. Sci., 1999, vol. 367, no. 6, pp. 406–407. 4. Sidorova, K.K. and Shumnyi, V.K., Genetika (Mos cow), 1999, vol. 35, no. 11, pp. 1550–1557. 5. Sidorova, K.K., Goncharova, A.V., Goncharov, P.L., and Shumnyi, V.K., S.Kh. Biol., 2012, no. 1, pp. 105– 109.
Translated by M. Bibov
GENERAL BIOLOGY
Symbiotic Mutants of Pea (Pisum sativum L.), an Important Genetic Source for Selection for Increased Nitrogen Fixation K. K. Sidorova and Academician V. K. Shumnyi Received October 31, 2013
DOI: 10.1134/S0012496614010190
hypernodulation genes (figure) [1–4]. Supernodulat ing mutant k301 marked by the gene nod4 was charac terized by abundant nodulation. It formed small nod ules all over the root throughout the vegetation period and demonstrated a high efficiency of nitrogen fixa tion. The mutant was characterized by a low seed yield compared to the initial cultivar Ramonsky 77. Super nodulating mutant k12 marked by the gene nod3 was induced from the cultivar Rondo. It also exhibited a high efficiency of nitrogen fixation and low productiv ity in comparison with the initial cultivar. The differ ence between these supernodulating mutants is that the mutant induced from the cultivar Ramonsky 77 had a fasciated stem, while the mutant obtained on the basis of the cultivar Rondo had a compact stem. The
An increase in the efficiency of the legume–rhizo bium symbiosis as an ecologically friendly and eco nomically inexpensive source of nitrogen taken up from the atmosphere is believed to be a challenging task of modern biology. In the symbiotic system, the macrosymbiont plays the role of an energy source, spending the products of photosynthesis not only on the production process (yield), but also on the forma tion of root nodules and nitrogen fixation. A microsymbiont consists of nodule bacteria that contain the enzyme nitrogenase, which reduces molecular nitrogen from the atmosphere to form ammonium. To increase the efficiency of nitrogen fix ation, selection may be carried out either at the level of the macrosymbiont via the development of legume genotypes characterized by a high productivity, intense nodulation, and nitrogen fixation or at the level of microsymbionts, the selection of which is aimed at the development of effective strains of nodule bacteria.
1
2
The goals of the present study were (1) the obtain ing and genetic study of a pea plant forms with improved symbiotic traits, such as active nodulation and nitrogen fixation and (2) the development of methods to use these forms for selection. The scientific novelty of this study is that we were the first to develop a method of the use of symbiotic mutants of pea plants for the selection aimed at the intensification of nitrogen fixation. The method is based on the combination of the alleles of different sym genes (the dominant hypernodulation gene Nod5 and the recessive supernodulation gene nod4) in the same genotype. Using the method of experimental mutagenesis, we obtained symbiotic mutants of pea plants, which were further used for identification of supernodulation and
Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, 630090 Russia
The root system of symbiotic mutant pea plants: 1, super nodulation; 2, hypernodulation.
59
60
SIDOROVA, SHUMNYI
Table 1. Productivity parameters (M ± m) of local endemic forms of pea plants from Syria and Egypt and the recurrent lines of the F5 generation obtained as a result of the following crossbreeding: , a recurrent line of the F7 generation marked with the supernodulation gene nod4, which was ob tained by the crossing of the cultivar Druzhnaya with the su pernodulating mutant k301 (nod4) × , a local endemic form Per plant Variant
number of seeds
weight of seeds, g
192.2 ± 3.1
36.5 ± 7.1
6.7 ± 0.8
218.7 ± 12.9 214.1 ± 10.4 254.6 ± 5.3* 194.8 ± 9.0 160.7 ± 4.5
65.7 ± 13.1* 40.5 ± 5.8 113.0 ± 12.1* 48.2 ± 5.8 43.8 ± 4.6
14.1 ± 2.9* 9.7 ± 1.5 16.8 ± 1.6* 8.0 ± 1.1 4.5 ± 0.3
height, cm Local endemic form from Egypt Recurrent lines: ** k6ed k7ed k12ed k26ed Local endemic form from Syria Recurrent lines: ** k2sd k11sd
205.6 ± 14.6* 89.1 ± 14.9* 13.2 ± 1.7* 190.0 ± 5.5* 54.2 ± 3.8 7.5 ± 0.5*
* Difference from the parental endemic form is significant at p < 0.05; ** the number of recurrent lines is given in accordance with the Catalogue provided by the Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences.
mutant carrying the dominant gene Nod5 was obtained on the basis of the cultivar Rondo. It is char acterized by hypernodulation and large nodules, which are located mostly at the upper part of the root. The mutant exhibited a high efficiency of nitrogen fix ation. The seed productivity of this mutant was higher
than that of the initial cultivar Rondo. We first identi fied the dominant gene Nod5 in the ancient pea plant cultivar Torsdag [4]. Currently, intense studies aimed at the develop ment of methods to use the symbiotic mutants for selection of pea plants in order to increase the effi ciency of nitrogen fixation are carried out. The use of the supernodulating mutant k301 (nod4), which was obtained on the basis of cultivars Ramonsky 77 and Torsdag and was marked by the gene Nod5, allowed us to develop the method of recurrent selection aimed at increasing in the efficiency of nitro gen fixation and high productivity. The method is based on the combining of different alleles of different genes within one genotype. The effectiveness of this approach was confirmed in experiments with feeder pea plant cultivars Druzhnaya and Novosibirskaya 1 [5]. Crossbreeding of local endemic forms of pea plants that originated from Syria and Egypt with the supern odulating mutant k301 (nod4) resulted in high levels of nodulation and nitrogen fixation in all recurrent lines of the F5 generation. However, all lines had a low seed yield. A positive result was achieved when the recur rent line of the F7 generation marked with the same supernodulation gene nod4 was used for crossbreeding instead of the mutant k301 (nod4). The line was obtained by crossbreeding of the feeder pea plant cul tivar Druzhnaya with the supernodulating mutant k301 (nod4) (Table 1). The results of the experiment showed that geno typic environment affected the expression of the supernodulation gene. Crossbreeding of two symbiotic mutants of pea plants, the hypernodulating mutant k74 (Nod5) and supernodulating mutant k12 (nod3), which were obtained on the basis of one cultivar Rondo, resulted in segregation of the F2 generation into supernodulat
Table 2. Symbiotic traits and productivity of supernodulating and hypernodulating mutants of pea plants (M ± m)
Variant
Symbiotic traits
Productivity
per plant
per plant
number of nodules
nitrogenase activity, nmole C2H4/h
height, cm
number of beans
number of seeds
Cultivar Rondo
138 ± 12
1269 ± 234
47.4 ± 0.7
6.6 ± 0.3
33.9 ± 1.8
Supernodulating mutant k12 (nod3)
802 ± 44
3166 ± 316
39.7 ± 0.8
5.4 ± 0.4
25.3 ± 1.5
Hypernodulating mutant k74 (Nod5)
380 ± 44
3425 ± 515
56.7 ± 2.1
11.5 ± 1.6
47.4 ± 7.2
Hypernodulating line obtained in re sult of the crossbreeding k74 × k12
1932 ± 29
8521 ± 480
69.4 ± 0.8
13.6 ± 1.6
67.4 ± 9.0
The results of the study show that both hyper and supernodulating mutants of pea plant can be used for selection aimed at the intensification of nitrogen fixation as a source for accumulation of nitrogen in soil. DOKLADY BIOLOGICAL SCIENCES
Vol. 454
2014
SYMBIOTIC MUTANTS OF PEA
ing and hypernodulating plants. The latter were char acterized by effective nodulation, active nitrogen fixa tion, and high productivity (Table 2). The results of our study demonstrate that symbiotic hyper and supernodulating mutant pea plants can be used in selection for an enhanced nitrogen fixation as a source of soil nitrogen accumulation. REFERENCES 1. Sidorova, K.K. and Uzhintseva, L.P., Dokl. Akad. Nauk, 1994, vol. 336, no. 6, pp. 847–849.
DOKLADY BIOLOGICAL SCIENCES
Vol. 454
2014
61
2. Sidorova, K.K. and Shumnyi, V.K., Dokl. Biol. Sci., 1997, vol. 353, no. 5, pp. 190–191. 3. Sidorova, K.K., Shumnyi, V.K., and Mishchen ko, T.M., Dokl. Biol. Sci., 1999, vol. 367, no. 6, pp. 406–407. 4. Sidorova, K.K. and Shumnyi, V.K., Genetika (Mos cow), 1999, vol. 35, no. 11, pp. 1550–1557. 5. Sidorova, K.K., Goncharova, A.V., Goncharov, P.L., and Shumnyi, V.K., S.Kh. Biol., 2012, no. 1, pp. 105– 109.
Translated by M. Bibov
