INCREASING PLANT TRANSFORMABILITY BY CYTOTYPE TRANSFER
20220174901 · 2022-06-09
Assignee
Inventors
Cpc classification
C12N15/8209
CHEMISTRY; METALLURGY
International classification
Abstract
A method of altering or transferring the cytotype of a plant line. In particular, transferring the cytotype of a transformation-recalcitrant plant line, e.g., a transformation-recalcitrant maize line, from a transformation-recalcitrant cytotype to a transformable cytotype so that the line becomes transformable while preserving its nuclear genome. The newly-transformable line may be produced using methods including backcrossing and/or haploid induction.
Claims
1. A method of increasing transformation efficiency rate in a recalcitrant maize line, comprising: a. obtaining a recalcitrant maize plant and collecting pollen therefrom; b. pollinating a recipient maize plant comprising normal A (“NA”) cytoplasm with pollen from the recalcitrant maize plant; and c. obtaining a progeny embryo therefrom; wherein the progeny embryo comprises the NA cytoplasm and at least the nuclear genome of the recalcitrant maize plant and wherein the progeny embryo possesses a higher transformation efficiency rate than the recalcitrant maize plant.
2. (canceled)
3. The method of claim 1, wherein the recalcitrant maize plant comprises a cytoplasm other than NA.
4. The method of claim 3, wherein the cytoplasm other than NA is selected from the group consisting of normal B (“NB”) cytoplasm, cytoplasmic-male-sterile C (“C” or “CMS-C”) cytoplasm, cytoplasmic-male-sterile S (“S” or “CMS-S”) cytoplasm, or cytoplasmic-male-sterile T (“T” or “CMS-T”) cytoplasm.
5. The method of claim 1 or 2, wherein the recipient maize plant is a haploid inducer plant.
6. The method of claim 5, wherein the haploid inducer plant is a maternal haploid inducer plant.
7. The method of claim 6, wherein the maternal haploid inducer plant comprises a mutated ig1 gene or a Matrilineal gene.
8. The method of claim 6, wherein the haploid inducer plant is a paternal haploid inducer plant.
9. The method of claim 6, wherein the paternal haploid inducer plant comprises a CENH3 mutation.
10. The method of claim 1, wherein the progeny embryo is grown into a progeny plant.
11. The method of claim 10, wherein the progeny plant is backcrossed with the recalcitrant maize plant for at least one generation.
12. The method of claim 11, wherein the progeny plant is the female parent in the backcross.
13. The method of claim 11, wherein the progeny plant maintains the NA cytoplasm.
14. The method of claim 11, wherein the progeny plant maintains the nuclear genome of the recalcitrant maize plant.
15. A method of increasing transformation efficiency rate in a recalcitrant plant line, comprising: a. obtaining a recalcitrant plant and collecting pollen therefrom; b. pollinating a recipient plant comprising transformable cytoplasm with pollen from the recalcitrant plant; c. obtaining a progeny embryo therefrom; and d. optionally i. growing the progeny tissue into a progeny plant; ii. backcrossing the progeny plant using the pollen from the recalcitrant plant for at least one crossing; and e. transforming tissue derived from the progeny embryo; wherein the progeny tissue comprises the NA cytoplasm and at least the nuclear genome of the recalcitrant plant and wherein the progeny tissue possesses a higher transformation efficiency rate than the recalcitrant plant.
16. A plant obtained by the method of claim 1.
17. A method of transforming a plant, comprising: a. obtaining a plurality of plant lines; b. testing for markers indicative of NA cytoplasm; c. selecting at least one line from the plurality of plant lines wherein the selected lines possess markers for NA cytoplasm; and d. transforming cells derived from the at least one selected line of step (c).
18. The method of claim 17, wherein the test detects a G nucleotide at a position that corresponds to position 11 of mitochondrial DNA sequence SEQ ID NO: 7.
19. The method of claim 18, wherein the test for markers indicative of NA cytoplasm detects the presence of a sequence corresponding to SEQ ID NO: 7.
20. The method of claim 17, wherein the test for markers indicative of NA cytoplasm comprises forward primer SEQ ID NO: 5 and reverse primer SEQ ID NO: 6, probe SEQ ID NO: 7 and/or probe SEQ ID NO: 8.
21. (canceled)
22. (canceled)
Description
DETAILED DESCRIPTION
[0058] One embodiment of the invention is a method of increasing transformation efficiency rate in a recalcitrant maize line, comprising: (a) obtaining a recalcitrant maize plant and collecting pollen therefrom; (b) pollinating a recipient maize plant comprising normal A (“NA”) cytoplasm with pollen from the recalcitrant maize plant; and (c) obtaining a progeny embryo therefrom; wherein the progeny embryo comprises the NA cytoplasm and at least the nuclear genome of the recalcitrant maize plant and wherein the progeny embryo possesses a higher transformation efficiency rate than the recalcitrant maize plant. Another embodiment is a method of conferring transformability to a recalcitrant maize line, comprising: (a) obtaining a recalcitrant maize plant and collecting pollen therefrom; (b) pollinating a recipient maize plant comprising normal A (“NA”) cytoplasm with pollen from the recalcitrant maize plant; and (c) growing a progeny plant therefrom; wherein the progeny plant comprises the NA cytoplasm and the nuclear genome of the recalcitrant maize plant and wherein the progeny plant is transformable. In one aspect, the recalcitrant maize plant comprises a cytoplasm other than NA. In another, the cytoplasm other than NA is selected from the group consisting of normal B (“NB”) cytoplasm, cytoplasmic-male-sterile C (“C” or “CMS-C”) cytoplasm, cytoplasmic-male-sterile S (“S” or “CMS-S”) cytoplasm, or cytoplasmic-male-sterile T (“T” or “CMS-T”) cytoplasm. In one aspect, the recipient maize plant is a haploid inducer plant, or more specifically a paternal haploid inducer plant. In another aspect, the paternal haploid inducer plant comprises a mutated ig1 gene or a CENH3 mutation.
[0059] In another embodiment, the progeny embryo is grown into a progeny plant. In one aspect, the progeny plant is backcrossed with the recalcitrant maize plant for at least one generation. In another aspect, the progeny plant is the female parent in the backcross. In yet another, the progeny plant maintains the NA cytoplasm and/or maintains the nuclear genome of the recalcitrant maize plant.
[0060] In yet another embodiment, the invention is a method of increasing transformation efficiency rate in a recalcitrant plant line, comprising: (a) obtaining a recalcitrant plant and collecting pollen therefrom; (b) pollinating a recipient plant comprising transformable cytoplasm with pollen from the recalcitrant plant; (c) obtaining a progeny embryo therefrom; and (d) optionally (i) growing the progeny tissue into a progeny plant; (ii) backcrossing the progeny plant using the pollen from the recalcitrant plant for at least one crossing; and (e) transforming tissue derived from the progeny embryo; wherein the progeny tissue comprises the NA cytoplasm and at least the nuclear genome of the recalcitrant plant and wherein the progeny tissue possesses a higher transformation efficiency rate than the recalcitrant plant.
[0061] Another embodiment of the invention is a method of transforming a plant, comprising: (a) obtaining a plurality of plant lines; (b) testing for markers indicative of NA cytoplasm; (c) selecting at least one line from the plurality of plant lines wherein the selected lines possess markers for NA cytoplasm; and (d) transforming cells derived from the at least one selected line of step (c). In one aspect, the test detects a G nucleotide at a position that corresponds to position 11 of mitochondrial DNA sequence SEQ ID NO: 7. In another aspect, the test for markers indicative of NA cytoplasm detects the presence of a sequence corresponding to SEQ ID NO: 7. Optionally, the test for markers indicative of NA cytoplasm further comprises forward primer SEQ ID NO: 5 and reverse primer SEQ ID NO: 6. Alternatively, the test for markers indicative of NA cytoplasm comprises probe SEQ ID NO: 7 and/or probe SEQ ID NO: 8, wherein the probes are differentially labeled with fluorophores.
[0062] These and other embodiments of the invention will be more fully understood in light of the following non-limiting examples.
EXAMPLES
Example 1. Cytotype Transformability and Cytotype Transfer
[0063] Markers to distinguish between NA, NB, and CMS cytoplasms were developed based on the NA and NB mitochondrial genomes disclosed by James O. Allen, et al., Comparisons Among Two Fertile and Three Male-Sterile Mitochondrial Genomes of Maize, G
TABLE-US-00003 TABLE 2 Transformation frequency of a selection of NA cytoplasm maize lines. Sum of Average of Total # Transformation Heterotic Variety Events Freq. Region Pool Line 1 0 0 Temperate Stiff Stalk (n = 1) Line 2 0 0 Temperate Stiff Stalk (n = 1) Line 3 2 0.32 Temperate Stiff Stalk (n = 2) Line 4 4 0.7 Temperate Stiff Stalk (n-2) Line 5 2 0.8 Temperate Stiff Stalk (n = 1) Line 6 13 1.3 Temperate Stiff Stalk (n = 4) Line 7 25 4.3 Temperate Stiff Stalk (n = 2) Line 8 25 6.25 Temperate Iodent (n = 1) Line 9 62 6.8 Temperate Stiff Stalk (n = 3) Line 10 610 50.1 Temperate Stiff Stalk (n = 3) Line 11 455 51.7 Temperate Stiff Stalk (n = 3) NP2222 315 31.8 Temperate Stiff Stalk (n = 5) Line 17* 0 0 Temperate Mixed (n = 1) Line 18* 34 6.8 Sub-Tropical Suwan (n = 2) *Lines 17 & 18 were tested in a second assay performed separately.
TABLE-US-00004 TABLE 3 NB lines and reciprocal crosses with NP2222. Average of Trans- Total formation Variety Cytotype Events Freq. Crossing Line 12 NB 0 0.0 group 1 Line 12 × NP2222 progeny NB 7 4.4 NP2222 × Line 12 progeny NA 9 4.1 Crossing Line 13 NB 0 0.0 group 2 Line 13 × NP2222 progeny NB 1 0.4 NP2222 × Line 13 progeny NA 3 0.8 Crossing Line 14 NB 9 4.5 group 3 Line 14 × NP2222 progeny NB 89 40.5 NP2222 × Line 14 progeny NA 92 46.0 Crossing Line 15 NB 0 0.0 group 4 Line 15 × NP2222 progeny NB 18 9.0 NP2222 × Line 15 progeny NA 23 10.3 Crossing Line 16 NB 0 0.0 group 5 Line 16 × NP2222 progeny NB 103 28.6 NP2222 × Line 16 progeny NA 71 22.5
[0064] In the crosses, the recipient line (i.e., female parent) is listed first and the pollen donor (i.e., male parent) is listed second. Without wishing to be bound by theory, it is believed that the NP2222 parent also imparts some other nuclear genetic factor that improves progeny transformability. Current transformation protocols are believed biased towards NP2222 and its derived lines. Even so, a 6% improvement (e.g., in crossing group 3) is a significant improvement in transformability, and a surprisingly good improvement at that.
[0065] By introducing the NA cytoplasm into the progeny, the transformation rate is significantly elevated in most lines compared to the recalcitrant parent having the NB cytotype. Even a modest increase in transformation rate, i.e., 0.0% to 0.8%, is a significant improvement over the nontransformability of the NB cytotype.
Example 2. Ig1-Mediated Cytotype Transfer
[0066] The maize mutant indeterminate gametophytel (ig1) produces maternal and paternal haploid progeny at 1-10% (Kindinger 1994). A NA transformable line was crossed by a heterozygous ig1 individual, PCR genotyped to identify heterozygous carriers and self-pollinated to create a NA version of the ig1 stock. F2 progeny were PCR genotyped and homozygous mutant ig1 individuals were pollinated by Line 13. One paternal haploid was identified and pollinated by the Line 13 recurrent parent. Transformation tests of the Normal B to Normal A were unsuccessful.
Example 3. No Detrimental Impacts on Yield
[0067] The effect of cytotype on hybrid yield was evaluated by reciprocally crossing Lines 7, 9, 10, 11 and NP2222 with Normal B testers and growing the progeny at approximately 10 yield trial locations. No obvious phenotypic differences were visible in the reciprocally crossed hybrid progeny and no statistically significant differences were observed across hybrids, locations or any of the interaction terms. This finding suggests that cytotype is an attribute that would not be expected to affect any obvious plant attribute and further exemplifies the significance of the differential effect on transformation competency.
[0068] Although the present invention has been described in considerable detail, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the versions contained herein.
[0069] All features disclosed in this specification may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.