CN105850737B - A kind of alpine rose " young beauty " chromosome doubling method - Google Patents

Abstract

The invention belongs to the field of biological breeding, and is specially used for the chromosome ploidy breeding of Rhododendron alpine "Pretty in Pink", in particular to a method for doubling the chromosomes of Rhododendron alpine "Pretty in Pink", which uses explants of Rhododendron alpine "Pretty in Pink" to induce callus , the callus is treated with the chemical reagent colchicine to double the chromosomes, and then transferred to the differentiation medium for culture to induce adventitious buds, and the adventitious buds are transferred to the proliferation medium for proliferation and culture, and then the strong seedlings are inoculated Carry out rooting culture, seedling hardening and transplanting in the rooting medium, observe chromosomes under a microscope, and screen out doubled plant seedlings. The present invention proposes a chromosome doubling method for the ornamental plant Rhododendron alpine "Pretty in Pink" for the first time. The polyploid alpine rhododendron "Pretty in Pink" bred by the invention can effectively improve the ornamental value of the alpine rhododendron "Pretty in Pink".

Description

A Chromosome Doubling Method of Alpine Rhododendron "Pretty in Pink"

technical field

The invention belongs to the field of biological breeding, and is specially used for the chromosome ploidy breeding of Rhododendron alpine "Pretty in Pink", in particular to a chromosome doubling method of Rhododendron alpine "Pretty in Pink".

Background technique

Alpine Rhododendron "Rhododendron Delavayi" (Rhododendron Delavayi "Cosmopolitan"), Rhododendron Delavayi "Cosmopolitan", native to the central and western regions of my country, evergreen shrubs and small trees, pink on the outside and white on the inside, with dense plants, stretched plant shape, leaves Green light. Polyploidy breeding is an effective breeding method and one of the important ways of plant evolution. "Giant type" is the most prominent external morphological feature of polyploids, showing thick stems, wide and thick leaves, rough leaf surfaces, darkened leaf color, large and thick corolla, increased petals, and rich colors. The advantages of flowering period and stress resistance enhancement of flower species increase the ornamental value and commodity value of flowers. With the increasing demand for this variety in the market, it is necessary to further innovate the germplasm resources of Alpine Rhododendron and cultivate new varieties.

Contents of the invention

In view of the above technical problems, the object of the present invention is to provide a technical method for doubling the chromosomes of Rhododendron alpine "Pretty in Pink". In order to achieve the purpose of the invention, the present invention provides the following technical solutions:

A method for doubling the chromosomes of Rhododendron alpine "Pretty in Pink", comprising the following steps:

(1) The explants of Rhododendron alpine "Pretty in Pink" were sterilized and transferred to the callus culture medium for culture to induce callus;

(2) using the chemical reagent colchicine to treat the callus to realize chromosome doubling;

(3) Transferring the callus treated with the chemical reagent colchicine to the adventitious bud medium for cultivation;

(4) transfer the differentiated adventitious buds to the proliferation medium and carry out proliferation culture;

(5) the strong and good tissue culture seedlings through the proliferation culture are inoculated in the rooting medium and carry out rooting culture;

(6) select the rooting seedling that root system grows to 2-3cm to carry out hardening, the mixed matrix that peat soil and perlite are made into;

(7) After hardening and transplanting, observe the number of chromosomes under a microscope, and screen out the plant seedlings with doubled chromosomes.

The method for explant disinfection described in step (1): process explant 30S with the alcohol of mass concentration 75%, rinse 4-5 times with sterile water, then process 1 with the mercuric chloride of mass concentration 0.1% minute;

The callus culture medium is prepared by the following method: add 30 g of sucrose, 6.5 g of agar powder, and 6 mg of 2-ip to 230 ml of WPM basic medium, adjust the volume to 1 liter with purified water, and adjust the pH value to 5.4, The culture temperature is 22±2°C, cultured in dark, and the culture time is 30 days.

The specific process of step (2) is: the callus is treated with colchicine with a mass concentration of 0.05% for 24 hours, and washed 4 times with sterile water;

The adventitious bud culture medium described in step (3) is prepared by the following method: add 30 g of sucrose, 6.5 g of agar, 0.1 mg of TDZ, and 1 mg of 2-ip to 230 ml of WPM basic medium; The pH value is 5.4, the culture temperature is 22±2°C, the light intensity is 3500 Iux, and the culture time is 45 days.

The proliferation medium described in step (4) is prepared by the following method: add 30 g of sucrose, 6.5 g of agar powder, 3 mg of 2-ip, and 0.5 mg of NAA to 230 ml of WPM basic medium, and dilute to 1 mg with purified water. 1, adjust the pH value to 5.4, culture temperature 22±2°C, light intensity 3500Iux, culture time 60 days.

Step (5) rooting medium, prepared by the following method: halve the macroelements in 230ml of WPM medium, that is, NH ₄ NO ₃ , (NH ₄ ) ₂ SO ₄ , MgSO ₄ .7H ₂ O in WPM medium , KH ₂ PO ₄ , KNO ₃ , K ₂ SO ₄ were reduced by half, 20g of sucrose, 6.5g of agar powder, 1g of activated carbon, and 0.8mg of IBA were added, and the volume was adjusted to 1 liter with purified water. The pH value was adjusted to 5.4, and the culture temperature was 22 ±2°C, light intensity 3500Iux, culture time 90 days.

The mixed matrix described in the step (6) comprises peat soil and perlite with a weight ratio of 5:1.

The specific process of step (7) is as follows: select the stem tip and root tip of the plant, make slices, and observe the chromosome doubling situation under a microscope.

The present invention provides a method for doubling the chromosomes of Rhododendron alpine "Pretty in Pink", which uses the chemical substance colchicine to treat the callus of Rhododendron alpine "Pretty in Pink" to induce polyploidy, and innovates the germplasm resources of Rhododendron alpine plants for use in Further cultivate new varieties and improve ornamental value.

detailed description

The technical scheme of the present invention is illustrated in conjunction with the examples, and the chemical substance colchicine is used to treat the callus of Rhododendron alpine "Pretty in Pink" to induce polyploidy.

Among them, the composition of 1 liter of WPM basic medium is:

Major elements: NH ₄ NO ₃ 400mg; (NH ₄ ) ₂ SO ₄ 132mg; MgSO ₄ .7H ₂ O 370mg; KH ₂ PO ₄ 170mg; KNO ₃ 400mg; K ₂ SO ₄ 900mg;

Calcium salt: CaCl ₂ 96mg; Ca(NO ₃ ) ₂ ·4H ₂ O 556mg;

Trace elements: H ₃ BO ₃ 6.2mg; MnSO ₄ .H ₂ O 22.3mg; Na ₂ MoO ₄ 2H ₂ O 0.25mg; CuSO ₄ .5H ₂ O 0.025mg; ZnSO ₄ .7H ₂ O 8.6mg;

Iron salt: Na ₂ -EDTA 37.3mg; FeSO ₄ .7H ₂ O 27.8mg;

Vitamins: inositol 100mg; niacin (vitamin PP) 0.5mg; pyridoxine hydrochloride 0.5mg; thiamine hydrochloride 0.5mg; glycine 2mg.

The specific method is as follows:

(1) Collection of explants

In spring, the young shoots grow actively and the weather is clear. In the morning, select the Alpine Rhododendron "Pretty in Pink" plant with good growth potential, cut off the young tissues and terminal buds at the top, take 30 explants, and peel off the part of the terminal buds covered. Scales, ready for sterilized processing.

(2) Disinfection treatment

First of all, each explant is brushed with a toothbrush dipped in detergent diluent, then rinsed with tap water for 30 minutes, treated with 75% alcohol for 30S on an ultra-clean bench, rinsed 4-5 times with sterile water, 0.1% chlorine Mercury treatment for 1 minute, then rinse with sterile water 4-5 times.

(3) Callus induced by explants

Inoculate the sterilized explants into the callus culture medium, add 30 g of sucrose, 6.5 g of agar powder, 2 mg of 2-ip, 0.5 mg of NAA, and dilute the purified water to 1 liter with 230 ml of WPM as the basic medium. Adjust the pH value to 5.4, culture at 22±2°C, and culture in dark for 30 days.

(4) colchicine treatment of callus

Under a sterile environment, the callus was soaked in 0.05% colchicine for 24 hours, and washed with sterile water for 4 times.

(5) Callus undergoes redifferentiation

The callus with granular texture and red color was selected and transferred to the redifferentiation medium. 230ml of medium is WPM basic medium, add 30g of sucrose, 6.5g of agar, 0.1mg of TDZ, 1mg of 2-ip, constant volume water to 1 liter, adjust pH value to 5.4, culture temperature 22±2℃, light intensity 3500Iux, culture The time is 45 days.

(6) Proliferation culture

The shoots grown from the callus after redifferentiation were transferred to the proliferation medium, and the medium of 230ml was based on WPM, adding 30g of sucrose, 6.5g of agar, 0.5mg of NAA, 2-ip 3mg, and distilled water to 1 1, adjust the pH value to 5.4, culture temperature 22±2°C, light intensity 3500Iux, culture time 60 days.

(7) Rooting culture

After proliferation and culture, select 2-3cm long sprouts and transfer them to rooting medium, 230ml of medium, of which NH ₄ NO ₃ , (NH ₄ ) ₂ SO ₄ , MgSO ₄ .7H ₂ O in WPM medium , KH ₂ PO ₄ , KNO ₃ , K ₂ SO ₄ are reduced by half, add 20g of sucrose, 6.5g of agar, 1g of activated carbon, 0.8mg of IBA, 0.3mg of IAA, dilute to 1 liter with purified water, adjust the pH value to 5.4, The culture temperature is 22±2°C, the light intensity is 3500 Iux, and the culture time is 90 days.

(8) Seedling hardening and transplanting

When the roots grow to 2-3cm in the rooting medium, the seedlings can be hardened and transplanted. Before transplanting, put the bottle seedlings in the greenhouse to acclimatize for about 7 days, open the bottle cap and place them for 3 days, take out the seedlings gently with tweezers, wash the medium with warm water, soak it in 1% carbendazim for 4 minutes, and plant it. Put it into the seedling tray, transplant the tissue culture seedling matrix ratio is peat soil:perlite is 5:1, pour water thoroughly, and use plastic film to shade from the sun for 3-5 days.

(9) Chromosome detection

Select the shoot tip and root tip of the Alpine Rhododendron "Pretty in Pink" plants treated with colchicine, observe the number of chromosomes through a microscope, and screen the doubled plants.

Claims (3)

1. A kind of 1. alpine rose " young beauty " chromosome doubling method, it is characterised in that comprise the following steps：

   （1）It is transferred in callus tissue culture base and is cultivated after alpine rose " young beauty " explant is sterilized, induction production Raw callus；

   The method of described explant sterilization：With the ethanol postincubation explant 30S of mass concentration 75%, with aseptic water washing 4-5 Time, then handled 1 minute with the mercury chloride of mass concentration 0.1%；

   Described callus tissue culture base, gained is prepared by following methods：Sucrose is added in 230ml WPM minimal mediums 30g, agar powder 6.5g, 2-ip 6mg, 1 liter is settled to pure water, adjusts pH value 5.4,22 ± 2 DEG C of cultivation temperature, light culture, Incubation time 30 days；

   Wherein 1 liter of WPM minimal medium composition is：

   A great number of elements：NH₄NO₃400mg；(NH₄)₂SO₄132mg；MgSO₄·7H₂O 370mg；KH₂PO₄170mg；KNO₃ 400mg；K₂SO₄900mg；

   Calcium salt：CaCl₂96mg；Ca(NO₃)₂·4H₂O 556mg；

   Trace element：H₃BO₃6.2mg；MnSO₄·H₂O 22.3mg；Na₂MoO₄·2H₂O 0.25mg；CuSO₄·5H₂O 0.025mg；ZnSO₄·7H₂O 8.6mg；

   Molysite：Na₂-EDTA 37.3mg；FeSO₄·7H₂O 27.8mg；

   Vitamin：Inositol 100mg；Nicotinic acid 0.5mg；Benadon 0.5mg；Thiamine hydrochloride 0.5mg；Glycine 2mg；

   （2）Callus is handled using chemical reagent colchicine, realizes chromosome doubling；

   Detailed process is：Colchicine processing 24h of the callus Jing Guo mass concentration 0.05%, with aseptic water washing 4 times；

   （3）Selection quality is in granular form, and the callus of rubicundity color, is transferred in adventitious bud culture base and is cultivated；

   Described adventitious bud culture base, gained is prepared by following methods：230ml WPM minimal mediums addition sucrose 30g, fine jade Fat 6.5g, TDZ 0.1mg, 2-ip 1mg, constant volume water adjust pH value 5.4,22 ± 2 DEG C of cultivation temperature, intensity of illumination to 1 liter 3500Lux, incubation time 45 days；

   （4）The adventitious bud differentiated is transferred in proliferated culture medium and carries out Multiplying culture；

   Described proliferated culture medium, gained is prepared by following methods：Sucrose 30g, fine jade are added in 230ml WPM minimal mediums Cosmetics 6.5g, 2-ip 3mg, NAA 0.5mg, 1 liter is settled to pure water, adjusts pH value 5.4,22 ± 2 DEG C of cultivation temperature, light According to intensity 3500Lux, incubation time 60 days；

   （5）Culture of rootage is carried out into root media by the good tissue culture plant inoculation of the stalwartness of Multiplying culture；

   Described root media, gained is prepared by following methods：In 230ml WPM minimal mediums, addition sucrose 20g, fine jade Cosmetics 6.5g, activated carbon 1g, IBA 0.8mg, 1 liter is settled to pure water, adjusts pH value 5.4；Wherein in WPM culture mediums NH₄NO₃、(NH₄)₂SO₄、MgSO₄、7H₂O、KH₂PO₄、KNO₃、K₂SO₄Amount reduces half；22 ± 2 DEG C of cultivation temperature, intensity of illumination 3500Lux, incubation time 90 days；

   （6）The rooted seedling that selection root system grows to 2-3cm carries out hardening, the mixed-matrix being made into turfy soil and perlite；

   （7）Chromosome Number Observation is carried out after acclimatization and transplantses under the microscope, filters out the plant seedling after chromosome doubling.
2. 2. according to a kind of alpine rose " young beauty " chromosome doubling method described in claim 1, it is characterised in that step Suddenly（6）Described in mixed-matrix, including weight ratio be 5:1 turfy soil and perlite.
3. 3. according to a kind of alpine rose " young beauty " chromosome doubling method described in claim 1, it is characterised in that step Suddenly（7）Specifically process is：The stem apex and the tip of a root of plant are selected, carries out section, observes chromosome doubling situation under the microscope.