The protocol for each assay reported below included Tripsacum-Z. diploperennis X corn hybrid lines and corn inbreds grown in growth chambers under controlled conditions at the Duke University Phytotron. The research design included three replicates of 128 plants per rep in a randomized block. Plants were grown in 4.5-inch diameter pots with nylon cloth covering the bottom of the pots to prevent larval escape out the holes in the bottom of the pots. Each plant was infested with 70 newly hatched first instar diapausing Western corn rootworm larvae at approximately three weeks post germination. The plants were harvested three weeks after infestation. The roots were carefully washed, then scored using the 1-6 Iowa rating scale (Hills and Peters ): 1 = no damage or only a few minor feeding scars; 2 = feeding scars evident, but no roots eaten off to within 1.5 inches of the plant; 3 = several roots eaten off to within 1.5 inches of the plant, but never the equivalent of an entire node of roots destroyed; 4 = one node of roots completely destroyed; 5 = two nodes of roots completely destroyed; 6 = three nodes of roots completely destroyed. Plants that have a root rating of 1 or 2 are resistant. After scoring the roots, resistant plants (i.e. those with a root rating of one or two) were repotted in 10-inch diameter pots and transferred to the greenhouse for backcrossing to corn to advance the recurrent selection breeding program for development of rootworm resistant corn lines. Leaf tissue from selected resistant plants was sampled for DNA fingerprinting to identify co-segregating molecular markers.
Bioassay #2 Twelve lines were tested in the second Phase II growth chamber insect bioassay at the Duke University Phytotron. These included five breeding lines (A, C, 97-3 X C, E, 97-1 X 97-3), one corn inbred (W64A), five lines in a Tripsacum cytoplasm (TC64, TC64 X TC, TC64 X 97-1, TC64 X A, and TC64 X 97-5). Results are summarized in Table 1.
Table 1. Results of Phase II Insect
|97-3 X C||18||6||3||2||9||4||0|
|97-1 X 97-3||29||10||2||4||14||9||0||0|
|97-3 X 97-1||23||2||2||3||10||8||0||0|
|TC64 X TC||13||5||1||3||5||4||0||0|
|TC64 X 97-1||25||10||1||4||5||13||0||2|
|TC64 X A||14||6||0||0||3||5||3||3|
|TC64 X 97-5||14||6||0||1||3||8||0||2|
After scoring, twenty resistant plants were repotted in 9-inch pots for cross pollinating with corn to advance the recurrent selection program for the development of rootworm resistant corn lines. The resistant families from this bioassay were A, C, 97-3 X C, E, 97-1 X 97-3, 97-3 X 97-1, TC64, TC64 X TC, and TC64 X 97-1.
Bioassay #3 Eleven lines were tested in the third Phase II growth chamber insect bioassay at the Duke University Phytotron. These included two corn inbred lines B73 and W64A, and nine breeding lines including 97-1 X 97-3, 97-1 X 97-5, 97-5 X 97-1, 97-5 X TC, 97-5, 97-1, 97-3, 97-3 X TC, 2023, 2019 X 2023, and E. The results are summarized in Table 2.
Table 2. Results of Phase II Insect
|97-1 X 97-3||32||6||0||1||12||14||5||0|
|97-3 X TC||7||3||0||0||3||4||0||0|
|97-1 X 97-5||31||4||0||0||4||19||7||1|
|97-5 X 97-1||30||6||0||0||8||14||7||1|
|97-5 X TC||30||6||0||0||7||17||5||1|
|2019 X 2023||7||4||0||0||2||3||1||1|
After scoring, three resistant plants were repotted in 9-inch pots and transferred to the greenhouse for cross pollinating with corn to advance the recurrent selection program for the development of rootworm resistant corn lines. The resistant families from this bioassay were 97-3 and E. In this bioassay with breeding lines that are now greater than 75% corn, resistance appeared to be lost in all but two lines. The two resistant lines were carried forward in the recurrent selection program, and the non-resistant lines were eliminated. These results raised the possibility that it may require more extensive backcrossing of corn lines to the resistant hybrids Tripsacorn and Sun Star than had been anticipated to effectively move the trait into corn.
Bioassay #4 Seventeen lines were tested in the fourth insect bioassay. These included Sun Dance hybrid families from earlier trials that had been backcrossed to corn, then self-pollinated, plus the corn inbred B73. Results are summarized in Table 3.
Table 3. Results of Phase II Insect
|9094 X 7009||30||7||23||0||7||19||3||1||0|
|7057 X 6088/6021||28||1||27||0||1||12||14||1||0|
|JY X 3029||22||6||16||0||6||9||4||0||3|
|3024 X W64A self||26||3||23||1||2||15||7||1||0|
|3024 X 6N615||28||1||27||0||1||15||9||3||0|
|97-5X97-1 BC B73||30||1||29||0||1||15||12||2||0|
|7099 X 7101||23||1||22||0||1||10||10||2||0|
|4021 X A188 self||24||1||23||0||1||8||8||5||2|
|3029 X A188 self||26||0||26||0||0||7||12||4||3|
|3028 X 6088||4||0||4||0||0||2||0||1||1|
|3028 X 7101||5||0||5||0||0||3||2||0||0|
|3028 X E||6||0||6||0||0||1||0||2||3|
|3024 X 7099||2||0||2||0||0||0||0||1||1|
Ten resistant plants were repotted in 9-inch pots for cross pollinating with corn to advance the recurrent selection program to develop rootworm resistant corn lines. In eleven of the families there was at least one resistant plant. The most promising families selected from this assay for further development in the breeding program were E (97-3-1X97-5-2), 9094 X 7009, and JY X 3029.
Bioassay #5 Ten lines were tested in the fifth bioassay. These included Sun Dance hybrid families from insect bioassay #4 that were crossed to corn then selfed or sib pollinated in the field during the summer of 1999. The lines were 99-1-2 (W64AXE), 99-2-3 (W64AXC), 99-15-4 (1056XW64A), 99-7-19 (7008XA188), 99-7-6 (7008XA188), 99-8-1 (7057XA188), 99-12-19A (7083A188), 99-12-5 X 99-12-3 (7083XA188), 99-16-3 (1091XW64A), and corn inbred W64A. Results are summarized in Table 4.
Table 4. Results of Phase II Insect
|99-12-5 X 99-12-3||54||30||24||14||16||12||12||0||0|
Fifty-one resistant plants were repotted in 9-inch pots and transferred to tall growth chambers for cross pollinating with corn to advance the recurrent selection program and for selfing or sibbing to increase seed quantities of these resistant families. All hybrid families in this bioassay exhibited resistance to Western corn rootworm. The most promising ones for the breeding program were (99-12-5 X 99-12-3), 99-12-19A, 99-7-19, and 99-16-3. Compare the roots of the resistant hybrid in Fig. 1 to the roots of a susceptible corn plant in Fig. 2. These F8BC6 breeding lines that are now greater than 90% corn confirm efficacy of moving the Western corn rootworm resistance trait from Tripsacum into corn using conventional breeding methods, and establish proof of concept for crop improvement by using the Tripsacum-Z. diploperennis genetic bridge to transfer Tripsacum genes into corn.
Figure 1. Roots of resistant hybrid.
2. Roots of susceptible corn.
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