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Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2010, № 5, p. 79-86. UDK 633.15:631.52/524
HAPLOINDUCTORS ISOLATION IN MAIZE: THREE CYCLES OF SELECTION ON HIGH FREQUENCY OF INDUCTION OF MATROCLINAL HAPLOIDS
O.A. Shatskaya
P.P. Lukyanenko Krasnodar All-Russia Research and Development Institute of Agriculture,
Russian Academy of Agricultural Sciences, Krasnodar-12 350012, Russia e-mail: o.shatskaya@mail.ru
Received August 26, 2009 S u m m a r y
With a using of hybridization of initial markers, revelation of individual plants with high frequency of induction and three cycles of selection during 14 generation of hybrid sample (РЕМ 48II x ЗМ 97)F1 the authors isolated maize haploinductors of ЗМК 1, ЗМК 1У and ЗМК 3 with high frequency of haploid induction — 5-7, 8-15 and 10-14 %, respectively. High effective haploinductors of ЗМК give an extended facility for using of haploidization method in breeding programs on isolation of maize homozygous lines.
Key words: Zea mays L., embryo marker, maternal haploids, haploid induction rate, inducers, selection.
The rapid method of creation of maize homozygous autodiploid lines from haploids obtained in vivo is the technique widely used in the world for past 15 years, as reported by literature data (1-3) and Web-sites of leading selection companies PIONEER (USA) (4) and Great Lakes Hybrids (USA) (5). This technique was supported by creation of effective haploid inducers for mass haploid production (1-3, 6-8) and improved methods for duplication chromosomes in haploid embryo cells (1, 8). Haploid sporophyte of matroclinal (maternal) type develops in unfertilized egg from embryo carrying gametal set of chromosomes (Zea mays L., n = 10). The fact of genetic control for haploids occurrence determined by both parental forms (both male and female) was shown by S.S. Chase (9) and many other authors (6, 10-12). In 1960ies, frequency of haploids in crosses with a well-known marker Purple Embryo Marker (PEM) amounted to 1,054 per 1000 examined caryopses, or 0,11%, while the line Stock 6 (discoverer - E.H. Coe) provided 3,23% haploids in progeny (10). The inheritance of high inducing ability by the hybrid (Stock 2 x Stock 6) FiBCi was demonstrated by K.R. Sarkar et al. (1972); after two cycles of testing and selection of hybrid material, haploid induction increased from 0,60 to 1,33%) (11). Self-pollination of the hybrid W 23 x Stock 6 has led to creation of WS 14 - the line inducing matroclinal haploids with frequency 2-5% (P. Lashermts and M. Beckert, 7). In 1990ies, new highly-effective haploid inducers based upon the markers PEM and Stock 6 were designed in various countries: ZMS 8 (Russia) - 3,8% (cited from 13), ZMK 1U (Russia) - 3-10% (1), MHI (Moldova) - 6,8% (2), RWS (Germany) - 8% (cited from 3). In this work, maize samples carrying the genes for coloration A1C1R1-nj:cudu are designated as “markers” and the forms able for haploid induction - “haploinducers”, or “inducers”.
The mechanism of induction of development an unfertilized egg into haploid embryo isn’t completely clear yet. Disturbances of microsporogenesis are assumed to be one of inducing factors. N.H. Jenaleeva et al. (13) performed embryological analysis of corn ovaries detected in 32-96 h after pollination with pollen from the inducer ZMS 8. It has been concluded that inducing ability of this line is provided by mutation leading to defective function of male gametes (immobility, inability for junction with female nuclei) and a consequent violation of double fertilization. Divisions in unfertilized egg can be stimulated by fertilization of only central cell of embryo sac and development of endosperm. S.T. Chalyk (2) has reported about high rates (11,0-14,7%) of microsporocytes in the variant with haploinducer MHI; it was assumed that aneuploid gametes result from violation of double fertilization.
Along with the method of labeled seedlings, identification of haploids can be performed by effective genetic marker system for color of dry caryopses including the genes A1C1R1-nj:cudu (developers - D.K. Nanda et al., 14). Transformation of haploids into diploid state is usually provided by treatment of apical meristems with aqueous solution of the alkaloid colchicine (S22N26O6). Duplicated set of chromosomes in nuclei of somatic cells leads to a consequent formation of viable gametes in some sectors of male and female inflorescences of a double haploid. The offspring from self-pollination of fertile plants is a new generation S1 autodiploid lines. Thus, it becomes possible to obtain homozygous lines during two field seasons including crossing of parental form and cultivation of hap-loids.
This work aimed at creation of highly-effective inducers of maize matroclinal haploids continues the study of haploidy phenomenon carried out since 1967 in the P.P. Luk’yanenko Krasnodar Research and Development Institute of Agriculture (KNIISKh) along the idea of academician M.I. Khadzhinov.
Technique. The experiment was carried out in 1984-2007 in the KNIISKh. In the first period, the initial material for hybridization were two groups of markers carrying the genes for coloration A1C1R1-nj - six synthetic populations created in the KNIISKh by M.V. Chumak (15) upon the basis of widely-used samples PEM and Stock 6, and three lines of embryo markers ZM 4, ZM 17, ZM 97 (related to the line ZMS 8) derived by selection in the Saratov State University (6). The initial material for estimation of inducing ability were the hybrids (PEM 48II x ZM 97)F2, [(8 RnjStock 6 x ZM 4) x ZM 97)]F1, (PEM 18 x ZM 17)F2, (5 RnjStock 6 x ZM 97)BC1, [(PEM 48IV x 8 RnjStock 6) x ZM 97]BC1, [(PEM 48II x 5 RnjStock 6) x ZM 97)]F2. For seed reproduction of inducers, self-pollination or sib mating (sibbing) in different generations were applied. Selection of inducers was performed according to the working program for a mass production of haploids; a large number of sources were used as maternal forms in early periods. The inducing ability of F1 hybrid markers was assessed upon results of crosses with different testers - hybrids and populations created with participation of inbred and autodiploid lines and races of maize from Latin America. F2 markers were estimated using the maternal forms - populations of 4th and 7th cycles of selection: BS 3, BS 16, RDA, RSSS-C Pr (kindly provided to KNIISKh by Dr. R.J. Lambert) (16). To identify the inducing ability of individual plants of markers, a single tester was used - the population BS 10 c7 (hereinafter BS 10), and in different years - several other populations and hybrids of a conventional semi-dent maize. In crosses with maternal testers, pollen from each marker the generations F1 and F2S0 was used to pollinate 10-15 corn-cobs on each maternal form, and 3-5 cobs were pollinated with pollen from individual plants the generations S0 and S1S0. Families of inducers the generations F6-F14 were evaluated by pollination of 4-10 cobs of the tester.
The individual and intra-family selection methods were used along with simultaneous assessment of inducing ability of selected samples. The samples were selected by phenotypic characteristics: manifestation degree of anthocyanin coloration of caryopses
and stem, amount of pollen in the panicle. To obtain haploids, the induction of development of unfertilized egg cell into haploid embryo under in vivo conditions was performed. The haploids were identified upon the method of genetic labeling with the genes A1C1R1-nj (the synthesis of anthocyanin resulting in colored stem, seed embryo and aleurone layer). The yellow-corn maternal forms of maize carrying recessive color determinant (acr genes) were pollinated with pollen of markers. Controlled crosses were carried out by manual pollination of plants along the common technique with isolation of cobs under parchment bags. The assumed haploids -caryopses with non-colored (white) embryos and anthocyanin-colored aleurone of endosperm - were harvested in the phase of dry corn. Hybrid caryopses with coloration of both embryo and endosperm were rejected. Corns from the group of assumed haploids were germinated in an incubator, and true haploids were identified by morphological features of 3-days-old seedlings (thickness of embryonic root, length of the 1st leaf). The frequency of haploids was determined as a percentage for the number of true haploids relative to the total number of labeled caryopses.
Results. Markers from Krasnodar - analogues of PEM and Stock 6 - have powerful habit of plants, but their inducing rates don’t exceed 0,10%, while the Saratov marker lines ZM demonstrated the rate of haploid induction up to 1,00% (a personal report of A.N. Zavalishina). In the first cycle of selection (C0 - zero cycle), hybridization of two groups of markers distinct by two main determinants has resulted in recombinant genotypes with valuable features of both parents (hybrid type of plant and increased ability to induction of haploids). The best combination was selected by tests of inducing ability in Fi and F2 hybrids. The average rate of haploid induction in eight of the studied markers Fi (0,06%) corresponded to that of parental forms, in six hybrids F2 (0,20%) - was within the values of ZM lines (Table 1). For test-crosses in Fi and F2 generations of hybrid markers, a mix of pollen collected from several plants of a plot was used. At the same time, some labeled cobs of maternal forms contained no caryopses with non-marked (haploid) embryo. The induction rate of the hybrid PEM 48II x ZM 97 generations F1 and F2 (respectively, 0,14 and 0,27%) significantly exceeded that of Saratov markers. Testing the hybrid (PEM 48 II x ZM 97)F1 on different maternal forms revealed the rate of haploid induction varying from 0,04% (the population of inbred lines) to 1,12% (autodiploid lines). In the variant with population of BS 10, 0,11% haploids relative the number of labeled caryopses were observed.
In 1986, the asessment of F2 hybrids have shown that five populations-analogues PEM and Stock 6 stimulated haploid induction with an average rate of 0,08%, three ZM lines - with the rate of 0,25%. For six hybrids F2S0 (zero cycle), the rate of induction averaged to 0,20% (395 haploids per 197 230 caryopses). However, in tests upon the population RDA, the hybrid (PEM 48II x ZM 97)F2 demonstrated highest inducing rate - 0,94% (53 haploids per 5670 caryopses). The unequal genotypic impact of RDA population on inducing ability of parental forms of this hybrid has been identified by the author. The rate of haploid induction in RDA population pollinated with marker’s pollen was high for this sample (0,14%, or 9 per 6460), and with pollen the line ZM 97 - medium (0,16%, or 3 per 1890). In crosses with three other testers (BS 3, BS 1916, rSsS-C Pr), the marker PEM 48II stimulated low rates (0,04% or 7 per 17 770), and the line ZM 97 - an increased rate (0,37%, or 59 per 15 890). A comparison of inducing rates obtained in crosses with different testers indicates different genetic and physiological nature of interaction of the genotypes PEM 48II and ZM 97 with genotype of a maternal form.
1. Efficiency, stages and methods of selection of maize markers ZMK 1 and ZMK 1U for high rates of induction of matroclinal hybrids
Year Inducer, initial sample (P), generation (F1-F14) and the cycle (С0-С3) of selection Number of examined samples, pcs. Rate of haploid induction (average for a range from-to), % System of reproduction, selection by each sample
1984 Populations — analogues PEM and 4 0,04 (0,02-0,06)a Reproduction and testing with
Stock 6 P Lines ZM R 3 0,08 (0,05-0,13) a the mix of pollen
1984 (Analogues PEM and Stock 6 x ZM)F^0 8 0,06 (0,03-0,14) a Reproduction and testing with
(PEM 48II x ZM 97)F1C0 1 0,14 the mix of pollen, identifica-
1986 (Analogues PEM, Stock 6 x ZM)F2C0 6 0,20 (0,11-0,27) a tion of the best genotype
(PEM 48II x ZM 97)F2C0 1 0,27
(PEM 48II x ZM 97)F2C0-S0C1 2 (of 10) 0,87; 2,14 Self-pollination, testing of pol-
1987 (PEM 48II x ZM 97)F3C0-S1C1 10 4,01 (2,35-6,92) a len from individual plants, selection of best plants
1988 (PEM 48II x ZM 97)F4C0-S2C1 4 6,22 (4,50-7,81) a Intra-family type of reproduction, testing by the mix of pollen, selection of best families
1989 ZMK 1 F5C0-S3C1 1 Combination of seeds from four best families, reproduction using the mix of pollen
1993 ZMK 1 F6C0-S4C1 1 7,53 Reproduction and testing by the mix of pollen, selection of best cobs
1994 ZMK 1 F7C0-S1C2 2 8,00b Intra-family type of reproduc-
1995 ZMK 1 F8C0-S2C2 7 5,64 (3,55-9,51) tion, testing with the mix of pollen, selection of best cobs
1996 ZMK 1U F9C0-S3C2 1 11,81 Combination of seeds from six best families, reproduction and testing by the mix of pollen
1997 ZMK 1U F10C0-S4C2 2 12,24 Intra-family type of reproduc-
1998 ZMK 1U F11C0-S1C3 2 12,19 tion, testing with the mix of
1999 ZMK 1U F12C0-S2C3 3 11,76 pollen, selection of best cobs
2001 ZMK 1U F13C0-S4C3 1 13,07
2002 ZMK 1U F14C0-S5C3 2 13,05
Note: a dash - determination of the parameter wasn’t performed, a - the range of induction rate for individual samples of markers - inducers, b - the rate of haploids was determined in one of two reproduced families.
It was hypothesized, that marker plants differ by presence and degree of manifestation of the induction genetic factor, so the next stage of selection was self-pollination and individual testing of pollen from the best hybrid (PEM 48II x ZM 97)F2S0 proposed as initial material for further selection . During such tests of pollen (i.e., the gametophyte generation S0S1) on the population BS 10, ten plants of the hybrid F2S0 showed the induction rates averaged to 0,41%. Two of these plants (№№ 2 and 3) demonstrated high rates - respectively, 0,87 and 2,14% (see Table 1), five plants - medium (from 0,15 to 0,40%), and three other plants revealed the ab-
sence of haploids in 15 labeled cobs. The variation of induction rates from 0 to 2,14% can be explained by presence or absence of the induction factor in plants of the hybrid population (PEM 48II x ZM 97)F2S0 (probably, owing to spontaneous mutation in genes that control microsporogenesis and/or microgametogenesis). High heritability of this factor was detected in all subsequent generations of the hybrid combination PEM 48II x ZM 97 - from F2 to F14, which caused a positive impact on efficiency of selection (see Table 1).
In contrast to the population PEM 48II, ZM lines and the marker (PEM 48II x ZM 97) showed kernel abnormalities in all generations starting from F3 - areas of undeveloped kernels on cobs ( cherezzernitsa), presence of corns having no embryos or feeble caryopses as a result of pollination by the mix of pollen. To maintain an optimal kernel content of cobs, self-pollination (F2 and F3) of marker samples was replaced in F4-F14 by intra-family pollination (i.e., by the mix of pollen from a plot); in some cases, a combination of both methods was used. Kernel abnormalities of cobs were also observed in maternal forms pollinated by inducers’ pollen (i.e., in marking crosses). During the reproduction of conventional maize populations and hybrids "within themselves", no abnormalities in kernel development were found.
The rate of haploid induction in 10 descendants of the plants 1 2 and 1 3 in the generation Si Si averaged to 4,01%. Four lines the generation S2S1 showed the rates 4,50-7,81% , and they became the basis for creation of the inducer ZMK 1 (embryo marker Krasnodar - zarodyshevyi marker Krasnodarskii). The rate of occurrence of matroclinal haploids in the population of dent corn P3 pollinated with pollen of the inducer ZMK 1 (S4C1) was 7,53% (140 haploids per1860 labeled caryopses). Therefore, the first cycle of selection for increased inducing ability performed in three generations (F2-F4) of the hybrid PEM 48II x ZM 97 has resulted in 28-fold raise of inducing rate compared with that in the initial form (F2C0 - 0,27%). The population ZMK 1 corresponds to a standard pollinator: plant height reaches 150-170 cm, branching panicle with good formation of pollen. Homozygous state of marker genes A1C1R1-nj in the inducer ZMK 1 provides clear coloration of corn in both inducer and hybrid caryopses of labeled cobs on maternal forms of dent and semi-flint type. However, in some lines of flint type and in the dent line T 22, the anthocyanin coloration of endosperm and embryo wasn’t manifested owing to presence in these forms of modifying genes or gene inhibitors.
Along with applying ZMK 1 for mass production of haploids, the selection of families for high inducing rate was continued. The second cycle of selection was performed in four seed generations of the inducer ZMK 1 (from F7C0-S1S2 to F10C0-S4S2) (see Table 1). Except the main characteristic (number of caryopses with haploid embryo detected in labeled cobs of maternal tester), the additional parameter was considered - kernel abnormalities (presence of grains without embryos, feeble caryopses, areas of undeveloped kernel) caused by violations in male generative sphere leading to a single fertilization or its complete absence. The frequency of haploid induction in seven families F8C0-S2S2 varied from 3,55 to 9,51%. The minimum number of corns without embryos (2,13%, or 13 defective corns per 623 examined) was detected in cobs produced by the family with lowest rate of haploid induction (3,55%). In turn, the family with highest (9,51%) inducing rate showed maximum proportion of such grains (11,4% or 31 per 302).
In 1996, a new population - the inducer ZMK 1U - was created (17). To obtain this sample, the mix of seeds taken in equal quantities from six families of the inducer ZMK 1 (F8C0-S2S2, inducing rates - from 3,55 to 9,51%) were sown. The plants grown from these seeds were pollinated with the mix of pollen collected from them, which resulted in the seeds F9C0-S3S2. When pollination of the maternal form BS 10 with the pollen of ZMK 1U, 11,81% matroclinal haploids were revealed (157 per 1330 labeled caryopses). The third cycle of selection in four generations (F11C0-S1S3-F14C0-S4S3) was aimed at stabilizing of high inducing ability of ZMK 1U. Seeds of different families were derived by both self-pollination and pollination with a mix of pollen considering the number of normally developed kernels in the cob.
The data of testing the pollen suggested a positive effect of intra-family selection of inducer’s cobs for high induction rate and the rate of germless seeds: the inducer ZMK 1U demonstrated stable high induction rates in four generations - from 11,76 to 13,07% (see Table 1) .
The obtained positive results (raise of haploid induction rates in the second cycle and stabilization - in the third cycle of selection) prove the correctness of considering an additional selection parameter - the proportion of germless seeds in inducer’s cobs. However, the analysis of long-term observations didn’t reveal a direct connection between these two parameters (the rate of haploid induction and the rate of germless seeds). At the same time, the presence of abnormal kernels (undeveloped embryos, areas on cobs without grains) should be expected when planning the resulting number of inducer’s. In 2002, it was recorded a small number of grains with normally developed embryo: in ZMK 1, ZMK 1U and ZMK 3 - respectively, 83, 85 and 41 grains per ear. The line ZMK 3 is an inbred offspring of the hybrid (3EM 48II x ZM 97) F2-S2S0, which had the best rate of haploid induction (7,81%). In this line generations F9-F12, the induction rate varied from 10,00 to 14,00% depending on environmental conditions and the genotype of source of haploids.
2. The rate and number of haploid caryopses in maize hybrids at different types of pollination with pollen from haploid inducers ZMK
Crossing Year Number, pcs. The rate of haploids, %
maternal forms labeled cobs | haploids average | from-to
Controlled (breeding nursery) Non-controlled (isolated plot) 2007 2004 8 22 270 1557 2864 11768 9,90 7,30-14,00 4,40 1,70-6,80
The use of haploid inducers ZMK makes possible a large-scale production of haploids by manual crossing with isolation of cobs in parchment packages (controlled pollination) and by open pollination of cobs on maternal plants with removed panicles performed on spatially isolated area (Table 2).
Significant variation of induction rates in the experiment and over the years was determined by genetic influence of both parents (male and female): different maize hybrids were used as sources of haploids, and pollinators were different samples from the working collection of ZMK inducers. Along with genetic factors, uncontrollable modifying environmental factors could affect induction rate as well. The planned amount of haploid corn can be produced by cultivation on large areas and by use of effective inducers.
The results of this study agree with conclusions of other authors about genetic determinants of haploidy phenomenon in maize. It is possible, that inducing ability of ZMK was inherited form genetic material the line Stock 6 present in genotypes of both parents of the marker PEM 48II x ZM 97. In 1999, F.K. Rober (18) using the method RLFP (restriction fragment length polymorphism) has obtained the data indicating probability of existence in ZMK of mutation distinguishing it from Stock 6. In the inducer KEMS (KEMS - Krasnodar embryo marker synthetic), only 21,10% RLFP-markers were similar with Stock 6, while 78,89% were distinct and indi-
cated the presence of X allele of an unknown parent or heterozygote. At the same time, the in induction rate of ZM 97 - the best on this parameter and a parent of ZMK - in different years didn’t exceed the upper limit of variation in rates exhibited by ZMS (2,23%) (6) and Stock 6 (3,23%) (10 ). A low (< 1 %) inducing ability of the second initial marker PEM 48II suggested a somewhat influence from the genotype of Stock 6 in this sample.
Almost 100-fold raise of inducing ability - from 0,14% in the hybrid (PEM 48II x ZM 97)FiS0 to 13,05% in the population ZMK 1U (Fi4C0-F4S3) can be explained by action of new mutation. Phenotypic manifestations of determinants reflecting violations in fertilization system of ZMK (areas of undeveloped kernels, germless seeds and feeble caryopses in the cob) indicate that this mutation can operate via disturbances of spermatogenesis, which was also observed in the experiments of N.H. Jenaleeva et al. (13) and S.T. Chalyk (2). The increased rates of haploid induction can be explained by several other factors. Maize has cross-pollination and the marker PEM 48II is a population, so it can be assumed the occurrence of recombinant genotypes carrying this mutation already in Fb and new combinations of genes - in F2. These genotypes were detected by double self-pollination in generations S0 and Si, and selected upon data of testing the pollen from individual plants. The unconventional types of pollination (sibbing and self-pollination) were performed to obtain the inducers’ seed progeny, could cause increased frequency of mutant allele or alleles and allelic interactions, i.e. changes in genetic and genotypic structure of the initial population (PEM 48II x ZM 97)F2. Finally, the pressure of longtime selection in 13 hybrid generations (F2-F14) probably contributed to raise in frequency of the mutant allele.
A high inducing potential of ZMK 1 was used in creation of the new inducer RWS by S. Deimling et al. (Germany) (8). According to their data, the rate of haploid induction (6,92%) in KEMS (ZMK 1) more than 3 times exceeded that of the line WS 14 (1,99%; France). The inducers ZMK 1, ZMK 1U and ZMK 3 are widely used in domestic and world selection practice for a mass production of maize haploids and rapid creation of homozygous lines. The introduction into ZMK of the marker gene for negative selection pehA providing synthesis of the enzyme monophosphate esterase was performed by USA researchers (19), which resulted in effective identification of haploids in culture of immature embryos.
So, three cycles of maize selection for high rate of haploid induction have resulted in creation of new inducers ZMK - lines and populations, which stimulate development of haploids with frequency of 6-13% and more. Using the inducers ZMK makes possible a mass production of maize haploids and a rapid creation of homozygous lines.
The study was performed under the guidance of Cand. Biol. Sci. V.S. Scherbak. The author would like to thank the staff of the N.G. Chernyshevsky Saratov State University, Doct. Biol. Sci. Prof. V.S. Tyrnov and Cand. Biol. Sci. A.N. Zavalishina, who kindly represented the initial samples of ZM lines, as well as the former KNIISKh employees Cand. Agric. Sci. E.R. Zabirova and Cand. Biol. Sci. M. V. Chumak for their assistance in experiments.
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