Pollen tube leading-in technique for tomato

A technology for pollen tube and tomato, which is applied to the field of tomato pollen tube introduction, can solve the problems that the transformation effectiveness cannot be fully explained, and the molecular detection cannot be carried out, and achieves the effects of avoiding disconnection of requirements, shortening breeding time, and avoiding comprehensive recombination.

Inactive Publication Date: 2002-04-03
SHANGHAI JIAO TONG UNIV
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AI Technical Summary

Problems solved by technology

Although the research on the pollen tube channel technology on tomato has been reported, molecular detection has not been carried out, and the performance of offspring of transformed plants has not been reported, which means that it cannot fully explain the effectiveness of transformation (Li Yuanxin et al., after pollination Application progress of DNA direct introduction technology in agricultural molecular breeding in my country, Beijing Agricultural Sciences, 1999, 17(2), 34-37), (Afp antifreeze protein gene (afp) introduced into tomato esterase and peroxidase Isoenzyme Analysis, Northern Horticulture, 1998, (1), 6~7)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] The recipient is the cultivar "Xianfeng", and the donor is the PBY520 plasmid with the target salt-tolerant gene HVA1. Select flower buds in the trumpet-mouth stage, isolate and bag them, and carry out artificial pollination the next day. 24 hours after pollination, use a razor blade to cut off 2 / 3 of the style in parallel, then use a 100 μl micro-syringe to draw 10 μl of donor DNA with a concentration of 0.615 / μl, and slowly insert it into the ovary along the style, as deep as 1 / 2 of the ovary. Gently lift the syringe up and carefully inject the donor DNA.

[0012] The fruit with the outer edge DNA will be reserved as a single fruit. The seedlings were sown separately in the next year, and the seedlings with four leaves (indicated by TG1) were irrigated with 150 mM NaCl, and the frequency of irrigation was 3 times a week for 4 consecutive weeks. The plants with strong salt tolerance were planted in the field to obtain 3 normal and strong plants. Then PCR detection w...

Embodiment 2

[0016] The recipient was the cultivar "Aihuang", and the donor was L. pennellii LA716. Select flower buds in the trumpet-mouth stage, isolate and bag them, and carry out artificial pollination the next day. 24 hours after pollination, use a razor blade to cut off 2 / 3 of the style in parallel, then use a 100 μl micro-syringe to draw 10 μl of donor DNA with a concentration of 0.667 / μl, and slowly insert it into the ovary along the style, as deep as 1 / 2 of the ovary. Gently lift the syringe up and carefully inject the donor DNA.

[0017] In the group numbered 324, a plant with green leaves and slow growth was found. This shows that the traditional law of leaf color inheritance also proves the feasibility and effectiveness of pollen tube technology in tomato.

Embodiment 3

[0019] The cultivar is "Xianfeng", and the donor is L. pennellii LA716. Select flower buds in the trumpet-mouth stage, isolate and bag them, and carry out artificial pollination the next day. 48 hours after pollination, use a razor blade to cut off 2 / 3 of the style in parallel, then use a 100 μl micro-syringe to draw 10 μl of donor DNA with a concentration of 0.715 / μl, and slowly insert it into the ovary along the style, as deep as 1 / 2 of the ovary. Gently lift the syringe up and carefully inject the donor DNA.

[0020] Since what is imported is the total DNA, the imported population is relatively large, and the offspring selection population is also large, so we use the first-order side branches of the TG1 generation plants to carry out cuttings in salt ponds (under substrate cultivation conditions, with 150mM NaCl stress), and select the ones with strong salt tolerance. The plants corresponding to the lateral branches.

[0021] The TG2 generation plants and the recipient v...

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Abstract

A pollen tube leading-in technique for tomato includes such steps as sleeving the alabastrum in horn stage in bag, 24-48 hr. after atificial pollination, truncating off 2/3 of style, using micro injector to suck DNA of donor, inserting it in ovary slowly up to half depth, and them carefully injecting the said DNA. It can lead in foreign DNA for the transfer of target gene. Its advantages are simple method and easily mastering it.

Description

Technical field: [0001] The invention relates to a tomato pollen tube introduction technology, which is used for cultivating new solanaceous varieties such as tomato, eggplant and pepper, and belongs to the technical field of crop breeding. technical background: [0002] Tomato is a strictly self-pollinating plant. Long-term artificial domestication and sexual hybridization have narrowed its genetic background. Some stress tolerance genes and quality genes are difficult to find in its cultivated species. According to some data, the salt tolerance of wild tomato relatives Lycopersicon peruvianum, L. cheesmanii, L. pennellii and L. pimpinellifolium is significantly higher than that of cultivated tomato. The Vc content of Peruvian tomato is 100 times that of cultivated tomato. The realization of gene transfer through sexual hybridization requires a long breeding cycle to achieve homozygosity of hybrids, and the close wild species have no cultivation value except for some resis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/02C12N15/09
Inventor 陈火英张建华陈云鹏庄天明
Owner SHANGHAI JIAO TONG UNIV
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