2n Occurrence of 2n-Pollen in Lilium
Hua-Ting, Chuang 2008/03/19 Outline Introduction Detection of 2n-pollen in Lilium
Conclusion Detection of 2n-pollen in Lilium Mechanisms of 2n-pollen in Lilium Utilization of 2n-pollen in Lilium Outline Introduction Detection of 2n-pollen in Lilium
Mechanisms of 2n-pollen in Lilium Utilization of 2n-pollen in Lilium Conclusion 2n-pollen (or unreduced pollen ) originate due to deviating meiosis in plants.
The process that leads to 2n-pollen formation is called meiotic nuclear restitution. Year Investigator Event 1927 Rosenberg Meiotic nuclear restitution was first proposed. 1982 Asano In lily, unreduced gametes (2n- pollen) are found in some wide interspecific hybrids. n-pollen 2n-pollen Metaphase Product Normal meiosis Plant 2n=2x=4
Meiotic nuclear restitution 2n-pollen (Ramanna and Jacobsen, 2003; Veilleux, 1985) Outline Introduction Detection of 2n-pollen in Lilium
Mechanisms of 2n-pollen in Lilium Utilization of 2n-pollen in Lilium Conclusion Detection of 2n-pollen in Lilium
Progeny Morphological Flow cytometry Microsporogenesis Meiotic observation The unexpected occurrence of polyploid progeny from 2x-2x, 4x-2x or 2x-4x hybridization has usually been the first indication of functional 2n-gametes. (Ramanna and Jacobsen, 2003; Veilleux, 1985) Percentage of aneuploids
Progeny Table 1.Chromosome number in progeny obtained by cro sses between Lilium Grand paradiso and 2n-pollen of L. Mona. Cross combinations Chromosome number Percentage of aneuploids Seed parents Pollen parents 24 40 41 42 43 44 45 46 47 48 Gran paradiso Mona 1 5 10 6 7 3 9 80.8 4x 2x 4x (Akutsu et al., 2007) Morphological 2n-pollenn-pollen Fig. 1. Comparison of n and 2n-pollen from Lilium Alaska (2n = 24) (a) pollen. (b) 2n pollen. Bar=100m . (Akutsu et al., 2007) Pollen grain diameter (m)
Morphological Pollen grain diameter (m) No. of pollen grains (a) (b) 3.5% 2n-pollen 28.1% 2n-pollen Fig. 2. Distribution of pollen grain size from Lilium inter specific hybrids . (2n = 24). (Van Tuyl et al., 1989) Flow cytometry (a) (c) (b) Number of nuclei Relative DNA content Fig. 3. Flow cytometric analyses of root tip and pollen from Lilium interspecific hybrids (a) Root (from lily 2n=2x=24) (b) Pollen (from lily 2n=2x=24) (c) Root (from lily 2n=4x=48). (Van Tuyl et al., 1989) Microsporogenesis (a) (b) Unbalanced chromosome distribution. This type of disjunction occurs the most commonly druing microsporogenesis in distantly related inter-specific hybrids. Unbalanced chromosome distribution. This type of disjunction occurs the most commonly druing microsporogenesis in distantly related inter-specific hybrids. Fig. 4. Microsporogenesis from Lilium interspecific hybrid (a) Triad shows a clear division of three nuclear formations by succe ssive cytokinesis (b) Sporads after tetrads stage, show dyads (D), triads (T) and tetrads. (Lim et al., 2004) Table +1. Frequency of types sporocytes at tetrad stage from Lilium hybrids.
Monad Dyad Triad Tetrad Others Total Revival 1(1)* 3(2) 124(97) 128(100) 8(5) 113(64) 18(10) 29(16) 176(100) 11(6) 97(53) 23(13) 45(25) 5(3) 181(100) Normal 2n-Pollen * The percentage is given in brackets. (Lim et al., 2004) Meiotic observation (a) (b) (c) Fig. 5. Meiosis and meiotic nuclear restitution from Lilium interspecific hybrid and (a) Balanced movement of homoeologous chromosomes at late ana phase I. ( ) (b) Unbalanced chromosome distribution. ( ) (c) Restitution nucleus formation in pollen mother cells without cytoki nesis (arrows; ). Unbalanced chromosome distribution. This type of disjunction occurs the most commonly druing microsporogenesis in distantly related inter-specific hybrids. Unbalanced chromosome distribution. This type of disjunction occurs the most commonly druing microsporogenesis in distantly related inter-specific hybrids. (Barba-Gonzalez et al., 2005 Lim et al., 2004) Meiotic observation 24 I 4II+16I
(1a) Metaphase I showing 3 bivalents (arrowheads) and 18 univalents. (a) (b) Fig. 6. Chromosome pairing at Metaphase I stage from Lilium interspecific hybrid (Barba-Gonzalez et al., 2004) Detection of 2-n pollen in Lilium
1.The unexpected occurrence of polyploid progeny from hybridization. 2.The larger (>90m) grains represent 2n pollen ( %). 3.2n-Pollen from diploid genotypes gave rise to1C and 2C peak by flow cytometry, can be related to haploid and diploid pollen. Detection 4.High frequency of dyads (53-64%) was formed after microsporogenesis. 5.Disturbed chromosome pairing. 6.Restitution nucleus formation in pollen mother cells without cytokinesis or unbalanced chromosome distribution. Outline Introduction Detection of 2n-pollen in Lilium
Mechanisms of 2n-pollen in Lilium Utilization of 2n-pollen in Lilium Conclusion Prophase I Metaphase I Anaphase I Cytokinesis I Metaphase II Anaphase II Cytokinesis II Product Normal FDR IMR SDR PMR FDR (Ramanna, 1979)FDR include mainly non-sister chromatids of each homologous pair of chromosomes. SDR the sister chromatid are included in the same gametes. Mother cell 2n=2x=4 Fig. 7. Schematic representation of the meiotic process and restitution mechanisms in microsporogenesis. (Lim et al., 2001; Lim et al., 2004; Karlov et al., 1998;Ramanna and Jacobsen, 2003; Veilleux, 1985) All univalent or a part univalent
All bivalent Fig. 8. Schematic representation of two possible types of meiotic nuclear restitution in a diploid interspecific hybrid (2n=2x=4) (a) FDR: First division restitution (b) SDR: Second division restitution. (Lim et al., 2001) A part of bivalents Bivalents Univalents Fig. 9. Genomic in situ hybridization to the chromosomes at Metaphase I stage from Lilium interspecific hybrids (L. longiflorum Asiatic, LA) for FDR Red fluorescence: Asiatic Yellow fluorescence: L. lolngiflorum Bar= 10 m. (Lim et al., 2001) All bivalents Fig. 10. Chromosome pairing at Metaphase I stage from Lilium interspecific hybrid (2n=24) for SDR. (Lim et al., 2004) Configurations / cells
Table 2. Frequency of chromosome association at metaphase I stage in night hybrids of Lilium. Hybrids No. of cells observed Configurations / cells Mean frequency 12II 11II 10II 9II 8II 7II 6II 5II 4II 3II 2II 1II 0II 0I 2I 4I 6I 8I 10I 12I 14I 16I 18I 20I 22I 24I Revial 125 115 8 2 11.8II+0.2I 99 95 3 1 11.9II+0.1I 118 108 6 4 32 5 12 3.7II+16.6I 87 13 20 28 10 2.1II+19.8I 882111 117 19 34 39 23 1.5II+21.0I 148 17 35 26 31 2.6II+18.8I 191 7 29 64 61 30 1.6II+20.8I 296 63 91 1.25II+21.49I 50 14 1.06II+21.88I Normal, II92% SDR, II92-96 % FDRII0% 2N-79SDRFDRIMRMIFDR2N-GAMETES FDRIMR (Van Tuyl et al., 2005) All univalent or a part univalent
A part bivalent or a part univalent Fig. 11. Schematic representation of three possible types of meiotic nuclear restitution in a diploid inter specific hybrid (2n=2x=4) (a) FDR: First division restitution (b) SDR: Second division restitution (c) IMR: Indeterminated meiotic restitution. (Lim et al., 2001) Configurations / cells
Table 2. Frequency of chromosome association at metaphase I in eight hybrids of Lilium. Hybrids No. of cells observed Configurations / cells Mean frequency 12II 11II 10II 9II 8II 7II 6II 5II 4II 3II 2II 1II 0II 0I 2I 4I 6I 8I 10I 12I 14I 16I 18I 20I 22I 24I Revial 125 115 8 2 11.8II+0.2I 99 95 3 1 11.9II+0.1I 118 108 6 4 32 5 12 3.7II+16.6I 87 13 20 28 10 2.1II+19.8I 882111 117 19 34 39 23 1.5II+21.0I 148 17 35 26 31 2.6II+18.8I 191 7 29 64 61 30 1.6II+20.8I 296 63 91 1.25II+21.49I 50 14 1.06II+21.88I FDR or IMR 2N-79SDRFDRIMRMIFDR2N-GAMETES FDRIMR (Van Tuyl et al., 2005) Table 3. Origin and parentage of Lilium interspecific hybrids and BC1.
2n-pollen Genome typea Accession number Female parent male parent F1 hybrids LA1~3 ,52,69 L. longiflorum Gelria Asiatic hybrid Whilito BC1 progeny ALA1 Asiatic hybrid ALA2~4 ,2,3 Asiatic hybrid Montreux ALA5~6 ,2 Asiatic hybrid Puccini ALA7~8 ,2 Asiatic hybrid Meribel a L and A, L. longiflorum and Asiatic hybrid, respectively. (Lim et al., 2001) Fig. 12. Triploid (2n=3x=36) chromosome constitution of BC (ALA1) lily, derived through the functioning of a 2n pollen that originated through IMR. 27A9L>5 (Lim et al., 2001) Table 4. Number of chromosomes and chromosome consti tution from Lilium interspecific hybrids and BC1. Genome typea Somatic chromosome number (2n) Chromosome constitutiona Number of cross-over break-points Number of recombinant chromosomes Nechanism of the 2n-gamete involved L A L/Ab A/Lb Total F1 LA1 24 12 LA2 LA3 BC1 ALA1 36 9 27 10 1 4 5 IMR ALA2 7 2 3 FDR ALA3 ALA4 48 ALA5 ALA6 ALA7 ALA8 odd IMR a L and A, L. longiflorum and Asiatic hybrid, respectively. b L/A and A/L, Recombinant chromosomes of L. longiflorum chromosome (centromere) with Asiatic chromosome and Asiatic hybrid chromosomeS (centromere) with L. longiflorum chromosome, respectively. (Lim et al., 2001) The chromosome of parent is even number.
Porphase I Metaphase I Anaphase I Cytokinesis I Metaphase II Anaphase II Cytokinesis II Product Normal FDR- lily type The chromosome of parent is even number. FDR IMR FDR (Ramanna, 1979)FDR include mainly non-sister chromatids of each homologous pair of chromosomes. SDR the sister chromatid are included in the same gametes. Mother cell 2n=2x=4 The chromosome no. of parent is odd. Fig. +1. Schematic representation of the meiotic process and restitution mechanisms in microsporogenesis. (Lim et al., 2001; Lim et al., 2004; Karlov et al., 1998;Ramanna and Jacobsen, 2003; Veilleux, 1985) FDR Failure of chromosome pairing (form all univalent or a part univalent) at meiotic I stage. Without the second division, abnormal cytokinesis and give rise to dyads instead of tetrads. 123 Equational division of sister chromatids. The chromosome of parent is even number (to compare IMR). SDR Normal chromosome pairing (All bivalent) at meiotic I stage.
Abnormal cytokinesis at meiotic II stage. Sister chromatids move to the same daughter cell. 123 IMR Display a mixture of FDR and SDR.
Failure of chromosome pairing (A part univalent) at meiotic I stage. A part sister chromosome move to the same daughter cell. The parental genomes are present in odd numbers. 123 Outline Introduction Detection of 2n-pollen in Lilium
Mechanisms of 2n-pollen in Lilium Utilization of 2n-pollen in Lilium Conclusion Utilization 2n-Pollen have been demonstrated to function in fertilization in many plants. 2 n (Veilleux, 1985) Fig. 13. 2n-Pollen germination from Lilium interspecific hybrid79418-2.
(Lim et al., 2004) Table 5. Pollen germination of n and 2n-gametes from Lilium Revial and interspecific hybrid Genotype Pollen type No. of pollen Germinated pollen Percentage In group In total Revial n 251 173 68.9 68.1 2n 3 All 254 130 41 31.5 19.1 85 54 63.5 25.1 215 95 44.2 Normal 2n-Pollen 2n-pollen (Lim et al., 2004) Chromosome recombinant
2n-pollenOA[BC12N-POLLEN] Fig. 14. Genomic in situ hybridization to the Chromosome at Post-Metaphase I stage from Lilium interspecific hybrid (with 2n-pollen) Post-Metaphase I stage in which sister chromatids of each chromo some are clearly visible, as are the recombinant chromatids in three pairs (arrow heads) and the double strand crossover event (arrows) Bar: 10m. (Barba-Gonzalez et al., 2005) Chromosome recombinant
27A9L>5 Fig. 15. Genomic in situ hybridization to the Chromosome from BC1 (ALA1) of Lilium (2n=3x=36). (Lim et al., 2001) Number of recombinant chromosomes
Table 6. Genome composition and number of recombinant chromo somes in progenies obtained from the cross of N2O trea ted OA lily hybrid to Asiatic (A) parent. Genotype Cross Ploidy Genome composition Number of recombinant chromosomes O (O/A) A (A/O) AAOA 3x 10 26(2) 2 4x 12(1) 36(1) 12 24 36 13 35 OAAA 1 (Barba-Gonzalez et al., 2006b) Table +2. Number of chromosomes and chromosome consti tution from Lilium interspecific hybrids and BC1. Genome typea Somatic chromosome number (2n) Chromosome constitutiona Number of cross-over break-points Number of recombinant chromosomes Nechanism of the 2n-gamete involved L A L/Ab A/Lb Total F1 LA1 24 12 LA2 LA3 BC1 ALA1 36 9 27 10 1 4 5 IMR ALA2 7 2 3 FDR ALA3 ALA4 48 ALA5 ALA6 ALA7 ALA8 IMR a L and A, L. longiflorum and Asiatic hybrid, respectively. b L/A and A/L, Recombinant chromosomes of L. longiflorum chromosome (centromere) with Asiatic chromosome and Asiatic hybrid chromosomeS (centromere) with L. longiflorum chromosome, respectively. (Lim et al., 2001) Despite these advantages, however, there is a limitation for routinely using 2n-pollen in Lilium breeding. Because the genotypes that produce 2n-pollen spontaneously are selected through laborious process. 1.2n- 2 n 2. 2 n 3. 2 n In order to overcome this difficulty, it would be essential to develop methods of inducing 2n-pollen. (Barba-Gonzalez et al., 2006) Table 7. The effects of heat treatment on Lilium OA hybrids.
Crossing number Parentage Pollen germination* (%) (range%) Oriental Hybrid Asiatic Hybrid Heated** Unheated Romero Star Connecticut King 31.4 (0-75) Bel Paso Gran Sasso 2.0 (n.a.) Pesaro 4.9 (0-10) 16.6 (0-100) Acapulco Sancerre 35.1 (0-90) 23.3 (0-60) Expression Au Revoir 25.0 (n.a.) Mero Star 2.6 (0-20) 7.6 (0-75) 0.53 (0-20) San Marco 37.5 (20-50) 6.0 (0-40) Bernini 1.9 (0-25) Tenerife Lanzorote 2.1 (0-30) Sissi Mirella 12.2 (0-60) 24.4 (0-75) Up 66% Up 7.0% *Plants of four normally sterile F1 hybrid OA genotypes were grown in a phytotron for 6 weeks and 2n-pollen 2n-pollen n.a. = not available; = genotype was not present. *The pollen germination percentage was scored counting only the large germinated 2n pollen grains. **10/8h30/8h under artificial lighting and 10/4h30/4h in the dark, creating day/night ratio of 16:8 h. (Lokker et al., 2005) Table 8. The effects of colchicine treatment on Lilium oriental.
Varieties Concentration (%) Flower buds of treament Percentage of death (%) Percentage of mutation (%) Con.Amore 0.00 300 0.0 0.02 12.7 0.05 37.3 4.7 0.10 49.3 9.5 0.20 81.3 1.2 Acapulco 5.7 2.3 16.3 5.0 33.3 25.8 47.0 11.3 *Lilium were treated by different concentration colchicine 2hen the young flower buds were about 0.5cm length for the production of 2n-gametes. The treating young flower buds were wrapped with a cotton wool soaked in colchicine and last for 3 days /14-18 2n-pollen2n-pollen *The treating young flower buds (0.5cm length) were wrapped with a cotton wool soaked in colchicine and last for 3 days. (Wu et al., 2007) Pollen grain diameter (m)
No. of pollen grains 2n-pollen N2O2N-POLLEN (N2O:O25:1)pressure was at 6 atm. Fig. 16. Distribution of pollen grain size from Lilium Lilies were treated with N2O gas for 24 h (a) Alaska (2n = 24) (b) Roma (2n = 24). (Akutsu et al., 2007) Fig. 17. Germination of 2n pollen from Lilium OA hybrid 951502-1
Fig. 17. Germination of 2n pollen from Lilium OA hybrid (a) Untreated (0 h treatment) (b) After 48 h of N2O treat ment. Bar: 0.5 cm. 2N (Barba-Gonzalez et al., 2006b) No. of ovaries developed Ploidy level of seeds (%)
Table 9. Interploidy crosses between diploid and tetraploid lily varieties and crosses between tetraploids and N2O treated pollen. Female Male No. of pollinated No. of ovaries developed No. of seeds obtained Ploidy level of seeds (%) patents Diploid Tetraploida Diploid diploid Roma Mona 5 305 (66) 100 (n=42) Tetraploid tetraploid Gran Paradiso Avignon 240 (48) 100 (n=18) Toreso 135 (27) 100 (n=30) N2O-treated pollen 11 8 175 (22) 100 (n=69) 4 3 19 (6) 100 (n=19) 17 (3) 100 (n=12) a Ploidy of mature seeds was examined by flow cytometry; n the number of seeds examined. (Akutsu et al., 2007) Table 10. Number of embryos obtained after N2O treatment of sterile F1 Oriental Asiatic (OA) lily hybrids and triploid BC (AOA) interspecific lily hybrids. Genotype Cross #pollinations #embryos 042923 AA* OA 19 179 042924 AA 3 35 4 1 6 28 052121 980072 AOA 14 052124 031028 20 10 32 950181 LD 2 051064 012046 N2OF12N-pollenBC1(2n-pollen) *AA = Asiatic, OA = F1 hybrid Oriental Asiatic, LD = L. longiflorum L. dauricum, AOA = Triploid hybrid obtained by the cross (Oriental Asiatic) Asiatic. (Barba-Gonzalez et al., 2006a) Pollen germination (Range %)
Table 11. Time effect on the pollen germination of diploid Asiatic (AA) and Oriental (OO) cultivars and diploid OA hybrids of Lilium 2n pollen producers. Code (Genome) Flower no. Pollen germination (Range %) Fresh 1-day old Pollyanna (AA) 12 95-100 Sorbonne (OO) 15 (OA) 17 85-100 0-