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CN-121975858-A - Sappan wood rooting genetic transformation system taking seeds as receptors

CN121975858ACN 121975858 ACN121975858 ACN 121975858ACN-121975858-A

Abstract

The invention provides a sappan wood rooting genetic transformation system taking seeds as receptors, and belongs to the technical field of biology. The invention provides a hematoxylin seed mediated genetic transformation method which comprises the steps of carrying out puncture treatment on hematoxylin seeds after peeling off seed coats, mixing an invasion solution containing a target gene with the hematoxylin seeds subjected to puncture treatment for infection, and planting the infected seeds to obtain transgenic hematoxylin. The invention screens and verifies the unique advantage of the seeds as the sappan genetic transformation receptor, establishes a set of sappan genetic transformation system which directly takes the complete seeds as the receptor for the first time, and successfully bypasses the key bottleneck of the traditional technology. The genetic transformation method remarkably improves the genetic transformation rate of sappan wood, the genetic transformation rate is 15%, and the genetic transformation rate is stable, and has the advantages of high efficiency and repeatability.

Inventors

  • HAN ZHIGANG
  • DING YUJIE
  • ZHANG JING
  • ZHANG YIXUAN
  • CHEN DONGHONG
  • SI JINPING

Assignees

  • 浙江农林大学

Dates

Publication Date
20260505
Application Date
20260407

Claims (10)

  1. 1. A method for sappan wood seed-mediated genetic transformation, comprising the steps of: Peeling off the seed coat of the sappan wood seeds, and performing puncture treatment to obtain the sappan wood seeds subjected to puncture treatment; mixing the invasion solution containing the target gene with the hematoxylin seeds subjected to puncture treatment for invasion to obtain infected seeds; planting the infected seeds to obtain the transgenic sappan wood.
  2. 2. The genetic transformation method according to claim 1, wherein the invading fluid comprises an infiltration fluid and a recipient bacterium comprising a gene of interest, and wherein the recipient bacterium comprises Agrobacterium rhizogenes.
  3. 3. The genetic transformation method according to claim 2, wherein the infiltration liquid comprises 9.5-10.5 mM 2-morpholinoethanesulfonic acid, 9.5-10.5 mM calcium chloride, 0.8-1.2 mM acetosyringone and 0.08-0.12% (v/v) tween 20, and the pH value of the infiltration liquid is 5.6-5.8; the agrobacterium rhizogenes comprises agrobacterium rhizogenes strain K599.
  4. 4. The genetic transformation method according to claim 1, wherein the puncturing treatment comprises: Inserting a needle from the position of the seed embryo axis near the embryo bud at the seed umbilicus end, and making a wound on the embryo bud; The depth of the needle is less than or equal to 5mm, the direction of the needle tip is inclined to the long axis of the seed, and the inclination angle is less than or equal to 30 degrees.
  5. 5. The genetic transformation method according to claim 1 or 4, wherein the infection is performed by intermittent negative pressure treatment, wherein the number of times of negative pressure treatment is 3, and each time the negative pressure is maintained at 0-4 KPa for 120s.
  6. 6. The genetic transformation method according to claim 1, wherein the seed coat is peeled off after washing the sappan wood seeds with running water for 10 to 14 hours, and the seed coat peeled off is washed with running water for not less than 4 hours before the puncture treatment.
  7. 7. The genetic transformation method according to claim 1, wherein the temperature of the cultivation after the seed is planted is 20-30 ℃ by covering 3-5 cm with soil.
  8. 8. The genetic transformation method according to claim 7, wherein the culturing is performed by culturing under dark conditions until the leaves are completely developed, and the light irradiation time of the light irradiation culturing is 16 h/day.
  9. 9. The genetic transformation method according to any one of claims 1 to 8, wherein the genetic transformation method is used for improving the expression stability of a target gene in sappan wood.
  10. 10. The use according to claim 9, wherein the gene of interest comprises CsOMT gene 80.

Description

Sappan wood rooting genetic transformation system taking seeds as receptors Technical Field The invention belongs to the technical field of biology, and particularly relates to a sappan wood rooting genetic transformation system taking seeds as receptors. Background Sappan wood (CAESALPINIA sappanl.) is a family of leguminous, genus Caesalpinia trees that are enriched in a variety of secondary metabolites with significant pharmacological activity, including brazilin, su Mutong a, etc. The active ingredients are proved to have various biological activities such as anti-inflammatory, antioxidant, antibacterial, anti-tumor, anti-neuroinflammation and the like by modern pharmacological researches, are widely applied in the field of traditional Chinese medicines, and also have huge application potential in the fields of modern medicines, functional foods, natural dyes and the like. However, the traditional sappan wood production mode mainly depends on cutting down arbor plants with growth period of up to several years and even more than ten years, the production mode is easy to lead to the reduction of wild sappan wood resources, aggravates ecological environment pressure, is difficult to meet increasing market demands, and forms a sharp contradiction between resource scarcity and market demand growth. Along with the development of molecular biology, analyzing the biosynthesis path of active ingredients of sappan wood, excavating key regulatory genes, and improving the content of the active ingredients by genetic improvement, has become an important direction of modern study of sappan wood. However, the traditional agrobacterium-mediated genetic transformation system faces serious challenges in the study of sappan wood, namely, the first genetic transformation system of sappan wood is not established, the second genetic transformation system of sappan wood is used as woody plant, secondary metabolites are rich, browning phenomenon in vitro tissue culture is serious, a regeneration system is extremely difficult to establish, and the genetic transformation under the conventional agrobacterium tumefaciens-mediated tissue culture condition is difficult to be suitable for the species. The lag of modern biotechnology research of sappan wood severely restricts sustainable utilization of sappan wood resources and efficient development of active ingredients thereof, and also restricts the possibility of improving the content of medicinal ingredients of sappan wood through modern biotechnology means. Disclosure of Invention Aiming at the defects in the prior art, the invention aims to provide a genetic transformation method of sappan wood, and the genetic transformation rate of sappan wood can be remarkably improved through the genetic transformation method. The invention aims at realizing the following technical scheme: the invention provides a sappan wood seed mediated genetic transformation method, which comprises the following steps: Peeling off the seed coat of the sappan wood seeds, and performing puncture treatment to obtain the sappan wood seeds subjected to puncture treatment; mixing the invasion solution containing the target gene with the hematoxylin seeds subjected to puncture treatment for invasion to obtain infected seeds; planting the infected seeds to obtain the transgenic sappan wood. Preferably, the infection liquid comprises an infiltration liquid and receptor bacteria containing target genes, wherein the receptor bacteria comprise agrobacterium rhizogenes. Preferably, the impregnating solution comprises 9.5-10.5 mM 2-morpholinoethanesulfonic acid, 9.5-10.5 mM calcium chloride, 0.8-1.2 mM acetosyringone and 0.08-0.12% (v/v) Tween 20, the pH value of the impregnating solution is 5.6-5.8, and the agrobacterium rhizogenes comprises agrobacterium rhizogenes strain K599. Preferably, the puncturing treatment method comprises the following steps: Inserting a needle from the position of the seed embryo axis near the embryo bud at the seed umbilicus end, and making a wound on the embryo bud; The depth of the needle is less than or equal to 5mm, the direction of the needle tip is inclined to the long axis of the seed, and the inclination angle is less than or equal to 30 degrees. Preferably, the infection is treated by intermittent negative pressure, the number of times of negative pressure of the intermittent negative pressure treatment is 3, and each negative pressure is maintained for 120 seconds under the condition of 4 KPa. Preferably, the sappan wood seeds are washed for 10-14 h in running water and then the seed coats are peeled off, and before the puncture treatment, the seed water from which the seed coats are peeled off is washed for more than or equal to 4h. Preferably, the soil is covered by 3-5 cm during seed planting, and the temperature of culture after planting is 20-30 ℃. Preferably, the cultivation mode comprises the steps of firstly cultivating under dark condition until the leaves are complete