LU103126B1

LU103126B1 - Virus-free seedling breeding method for jinsha pomelo and application

Info

Publication number: LU103126B1
Application number: LU103126A
Authority: LU
Inventors: Jialiang Zhou
Original assignee: Univ Jinggangshan
Priority date: 2023-05-17
Filing date: 2023-05-17
Publication date: 2023-11-30

Abstract

The present invention belongs to the technical field of fruit tree planting, and particularly relates to a virus-free seedling breeding method for Jinsha pomelo and application. According to the present invention, by limiting types and concentrations of disinfectants, disinfection time, picking time, length and a grafting method of stem segments of Jinsha pomelo, stem tips with 3-4 leaf primordia reserved are taken as scions, the scions are directly grafted onto rootstocks by a stab-inoculation method without disinfection treatment, and grafted seedlings obtained by grafting can be cultured to greatly reduce a pollution rate of grafting, improve a survival rate and a germination rate of grafting, and realize virus-free seedling breeding of Jinsha pomelo.

Description

VIRUS-FREE SEEDLING BREEDING METHOD FOR JINSHA POMELO AND HU108726

APPLICATION TECHNICAL FIELD

[01] The present invention belongs to the technical field of fruit tree planting, and particularly relates to a virus-free seedling breeding method for Jinsha pomelo and application.

BACKGROUND ART

[02] At present, there are some problems in the development of Jinsha pomelo industry, such as variety aging and degradation, low yield, poor fruit quality and low benefit. Due to the strong market demand for seedlings, in order to win as many orders as possible, most seedling raising units manufacture in a rough and slipshod way in the process of seedling raising, and uses manual operation, and backward seedling raising methods, which causes low work efficiency, low seedling raising ability and unqualified seedling quality; and the market seedling raising does not pay attention to the prevention and control of viral diseases and other dangerous pests and diseases, and a large number of diseased seedlings are popular, which endangers the sustainable development of Jinsha pomelo industry, and breeding virus-free Jinsha pomelo seedlings is particularly urgent.

SUMMARY

[03] An objective of the present invention is to breed virus-free seedlings of Jinsha pomelo with a high transplanting survival rate, a high germination rate, a low pollution rate and a strong antiviral ability.

[04] A virus-free seedling breeding method for Jinsha pomelo is provided in the present invention and includes: picking semi-lignified tender shoots of Jinsha pomelo from March to May, and cutting the tender shoots to 1-3 cm to obtain stem segments of

Jinsha pomelo;

[05] performing first disinfection on the stem segments of Jinsha pomelo with 1 mercuric chloride having a concentration of 0.1 wt.% or sodium hypochlorite having a HU108726 concentration of 5 wt.%, performing second disinfection with the sodium hypochlorite having a concentration of 5 wt.%, and taking the stem tips with 3-4 leaf primordia reserved as scions;

[06] grafting the scions onto rootstocks by a stab-inoculation method to obtain grafted seedlings for culture;

[07] the stab-inoculation method includes: under aseptic conditions, removing tops of the rootstocks, to reserve stems having a length of 1.5-2 cm, longitudinally cutting the rootstocks at positions 1 cm away from top ends, horizontally turning the rootstocks by 180 degrees, longitudinally cutting the rootstocks at positions 1 cm away from top ends, connecting the two incisions, cutting through xylem and phloem at one third of stem segments of the rootstocks to form rectangular incisions, and grafting the scions into the rectangular incisions.

[08] According to the present invention, by limiting picking time and stem segment length of the semi-lignified tender shoots of Jinsha pomelo, types and concentrations of disinfectants, disinfection time, and a grafting method of Jinsha pomelo, the stem segments of Jinsha pomelo having a length of 1-3 cm is sequentially disinfected with the mercuric chloride having a concentration of 0.1 wt.% and the sodium hypochlorite having a concentration of 5 wt.% for 10-12 min, after disinfected materials are cultured, the stem tips with 3-4 leaf primordia reserved are taken as the scions, the stem tips with 3-4 leaf primordia reserved do not need to be disinfected anew, the scions are directly grafted onto the rootstocks by the stab-inoculation method, and the obtained grafted seedlings may be cultured to greatly reduce the pollution rate of grafting, improve a survival rate and a germination rate of grafting, and realize virus-free seedling breeding of Jinsha pomelo.

BRIEF DESCRIPTION OF THE DRAWINGS

[09] In order to describe the examples of the present invention or the technical solutions in the prior art clearer, and the accompanying drawings required by the examples are briefly described below. 2

[10] FIG 1 shows test results of disinfection and germination of stem segments in HU108726 different seasons.

[11] FIG 2 is a schematic diagram of four grafting methods; A is "T" grafting, B is "A" grafting, C is cleft grafting and D is a stab-inoculation method.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[12] A virus-free seedling breeding method for Jinsha pomelo is provided in the present invention and includes: picking semi-lignified tender shoots of Jinsha pomelo from March to May, and cutting the tender shoots to 1-3 cm to obtain stem segments of

Jinsha pomelo.

[13] Disinfecting the stem segments of Jinsha pomelo for 10-12 min with mercuric chloride having a concentration of 0.1 wt% and sodium hypochlorite having a concentration of 5 wt.% for 10-12min to obtain first disinfected stem segments. The disinfection way is oscillation and/or shaking.

[14] Disinfecting the first disinfected stem segments for 10-12 min with the mercuric chloride having a concentration of 0.1 wt% for 10-12 min to obtain disinfected materials. The disinfection way is oscillation and/or shaking.

[15] Culturing the disinfected materials, and taking stem tips with 3-4 leaf primordia reserved as scions. Culture temperature is 262°C, a photoperiod is 16 h/d, and light intensity is 2500 Ix. A culture medium used for culture takes an MT solid medium or an MS solid medium as a basic culture medium, and also includes 1.0 mg/L 6-BA and 40 g/L of sucrose, with pH of 5.8-6.0.

[16] Grafting the scions onto rootstocks by a stab-inoculation method to obtain grafted seedlings. In the present invention, a preparation method for the rootstocks includes: peeling off inner and outer seed coats of Fructus Aurantii seeds under aseptic conditions, performing sterilization, to obtain rootstock seeds, inoculating the rootstock seeds into a solid culture medium, and performing culture o obtain the rootstocks. In the present invention, a bactericide used for sterilization includes the sodium hypochlorite having a concentration of 5 wt.%, sterilization time is 5-10 min, and a sterilization way includes soaking. In the present invention, the solid culture medium takes the MT solid 3 medium or the MS solid medium as a basic culture medium, and also includes 40 g/L of HU108120 sucrose. À culture way is dark culture, temperature is 26£2°C and time is 21 d. Before grafting, pretreating the rootstocks under a condition of light intensity of 2000 Ix for 1-2 d.

[17] Culturing the grafted seedlings to obtain virus-free seedlings of Jinsha pomelo.

Culture temperature is 26+2°C, a photoperiod is 16 h/d, and light intensity is 2500 Ix. A culture medium used for culture takes the MS solid medium as a basic culture medium, and also includes 40 g/L. of sucrose, with pH of 5.8-6.0. In the present invention, the grafted seedlings are cultured until 2 to 3 true leaves grown, then transferred to a new culture medium for continuous culture, and when 4 to 5 true leaves grown, the grafted seedlings are transplanted into a culture bowl for continuous culture; and a culture medium in the culture bowl is composed of fine sand, vermiculite and peat soil according to a volume ratio of 1:1:1.

[18] The present invention further provides application of the breeding method in the technical solution in cultivating excellent varieties of Jinsha pomelo.

[19] In order to further describe the present invention, the technical solutions provided by the present invention will be described in detail below with reference to accompany drawings and examples, but they cannot be understood as limiting the scope of protection of the present invention.

[20] Example 1

[21] (1) In late March, May, July, September and October, semi-lignified tender shoots of Jinsha pomelo with a life cycle within triennial were picked from Jinggang honey pomelo breeding base in Jinggangshan agricultural high-tech industrial demonstration zone separately, and cut into stem segments have a length of about 5 cm, each stem segment had 2-3 axillary buds, the stem segments were washed in a detergent diluted by 100 times, then rinsed with tap water for 30 min, placed on an ultra-clean workbench, repeatedly rinsed with sterile water for several times and put into a sterile beaker for later use; and

[22] (2) the stem segments treated in step (1) were disinfected by oscillation and shaking with sodium hypochlorite having a concentration of 5 wt.% for 10 min, rinsed 4 by oscillation with high-temperature sterilized water for 3 times, then disinfected by HU108726 oscillation and shaking with the sodium hypochlorite having a concentration of 5 wt.% for 10 min, rinsed by oscillation and shaking with the high-temperature sterilized water for 3 times, inoculated into an MT solid culture medium, and cultured at 26+2°C, a photoperiod of the culture was 16 h/d, and light intensity was 2500 Ix. 30 stem segments are treated each time and repeated three times, after 10 d of culture, a pollution rate is counted, after 30 d of culture, a germination rate is counted, results show that the stem segments picked in October has the highest average pollution rate and the lowest germination rate. The stem segments picked from March to May have a low pollution rate and a high germination rate, and the stem segments picked in May has the lowest pollution rate and the highest germination rate (FIG 1).

[23] Example 2

[24] The semi-lignified tender shoots picked in late May and cut into the stem segments have a length of 5 cm in Example 1 are used as experimental materials, the stem segments are treated in the same way as in Example 1, the only difference is that before inoculation, some stem segments are cut into stem segments have a length of 1 cm, 2 cm, 3 cm and 4 cm, and inoculated into a culture medium (an MT solid medium+1.0 mg/L. 6-BA+40 g/L sucrose) for culture, 20 stem segments are treated in each group, each group is repeatedly treated for 3 times, after 10 d of culture, a pollution rate is counted, after 30 d of culture, a survival rate is counted, results show that the survival rate is 8.3% and the pollution rate is 88.3% when the stem segments have a length of 5 cm; and when the stem segments have a length of 3 cm, 2 cm and 1 cm, the pollution rate is greatly reduced, the survival rate is slightly improved, the pollution rate is the lowest when the stem segments have a length of 1 cm, and an effect is good when the stem segments have a length of about 1-3cm (Table 1).

[25] Table 1 Disinfection test results of stem segments having different sizes (average value + standard error)

[26] Note: Different letters in the table indicate a significant difference between two treatments, and p = 0.01.

[27] Example 3

[28] (1) in late May, semi-lignified tender shoots of annual Jinsha pomelo were picked from Jinggang honey pomelo breeding base in Jinggangshan agricultural high-tech industrial demonstration zone, and cut into stem segments have a length of about 5 cm, each stem segment had 2-3 axillary buds, and the stem segments were washed according to the step (1) in Example 1 and cut into stem segments have a length of 2 cm and each contain a bud, which were used as stem segments of explants; and

[29] (2) according to the way in Table 2, the stem segments of the explants were disinfected differently, inoculated into an MT solid medium separately, and cultured according to the conditions in Example 1, 20 stem segments were treated in each group, each group was repeatedly treated for 3 times, after 10 d of culture, a pollution rate was counted, after 30 d of culture, a survival rate was counted, results showed that browning plants appeared in different disinfection treatments, the largest number of browning plants appeared in treatment D, and the highest survival rate appeared in treatment C after disinfection. The pollution rates in treatments E, D and B were higher than that in treatment C.

[30] Table 2 Disinfection methods and test results

First disinfection Second disinfection Pollution rate browning (%) (%0)

Mercuric Sodium

Treatment chloride having |10 min [hypochlorite |10min |3 33.3+2.9c 50.0+5.0a 6

: 0703126 of 0.1 wt.% concentration of 5 wt.%

Mercurie

Mercuric chloride

Treatment | chloride having 10 min [having a|10min |2 43.3+7.7bc [30.0+5.0b

B a concentration concentration of 0.1 wt.% of 0.1 wt.%

Sodium Sodium hypochlorite hypochlorite

Treatment having a|10 min |having a|10 min 1 38.3+2.9bc |55.0+5.0a

C concentration of concentration wt.% of 5 wt.%

Mercunc

Treatment | chloride having min |/ / 4 45.0+8.7b 18.3+2.9c

D a concentration of 0.1 wt.%

Sodium hypochlorite

Treatment having a|20 min |/ / 3 60.0+5.0a 25.0+5.0bc

E concentration of 5 wt.%

[31] Note: After each treatment is disinfected, the stem segments are rinsed by oscillation and shaking with high-temperature sterilized water for 3-4 times.

[32] Example 4

[33] 1. Preparation of rootstocks for grafting

[34] 1) After fruits of Fructus Aurantii matured, seeds were peeled off, soaked in alcohol having a concentration of 75 wt.% for 1 min, washed with high-temperature sterilized water for 3 times, and inner and outer seed coats were peeled off on a sterilized glass plate; 7

[35] 2) the treated seeds were sterilized with sodium hypochlorite having a HU108726 concentration of 5 wt.% for 5-10 min on an ultra-clean workbench, rinsed with the high-temperature sterilized water for 5 times, and inoculated into a culture medium (an

MT solid culture medium+40 g/L of sucrose) under aseptic conditions. 4 seeds of

Fructus Aurantii were inoculated into each test tube, a variety and date were marked, and the seeds were put into a culture room at 26+2°C for 21 d to obtain etiolated seedlings; and

[36] 3) the etiolated seedlings were placed in the culture room for 1-2 d, and light hardening was performed on the etiolated seedlings with intensity of 2000 Ix to obtain grafting rootstock.

[37] 2. Shoot-tip grafting

[38] 1) In the late April to late May of the year when Jinsha pomelo was planted, during a growth period of spring shoots, summer shoots and autumn shoots, tender shoots of Jinsha pomelo were collected and put into a clean fresh-keeping bag before being brought back to a laboratory; the tender shoots of Jinsha pomelo were cut into stem segments have a length of about 2 cm, soaked in a detergent diluted by 100 times for 5-10 min, and then rinsed with tap water for 30 min to remove dust, the washed tender shoots were put on an ultra-clean workbench, rinsed repeatedly with sterile water at first, and finally put into a sterile beaker as spare materials;

[39] 2) the stem segments prepared in step 1) were treated as follows: the stem segments were disinfected by oscillation and shaking with sodium hypochlorite having a concentration of 5 wt.% for 10 min, rinsed by oscillation with high-temperature sterilized water for 3 times, then disinfected by oscillation and shaking with the sodium hypochlorite having a concentration of 5 wt.% for 10 min, rinsed by oscillation and shaking with the high-temperature sterilized water for 3 times, finally inoculated into an

MT solid culture medium for culture, culture conditions: culture temperature was 26+2°C, a photoperiod was 16 h/d, and light intensity was 2500 1x;

[40] 3) on the ultra-clean workbench, rootstock seedlings with thick stems were selected from the rootstocks which had been subjected to light hardening for 1 d in step 1, small parts at top ends and roots were cut off to obtain rootstocks having a stem 8 length of about 2-3cm and a root length of about 3-4 cm; HU108120

[41] 4) 40 plants were grafted by a "T" grafting method, a "A" grafting method, a cleft grafting method and a stab-inoculation method (FIG 2) separately, in which specific steps of the stab-inoculation method are as follows, and the other grafting methods use conventional steps:

[42] the stab-inoculation method: under aseptic conditions, tops of the rootstocks are removed to reserve stems having a length of 1.5-2 cm, the rootstocks were longitudinally cut at positions 1 cm away from top ends, the rootstocks were horizontally turned by 180 degrees, the rootstocks were longitudinally cut at positions 1 cm away from top ends, the two incisions were connected, xylem and phloem at one third of stem segments of the rootstocks were cut through to form rectangular incisions, under a high-power stereomicroscope, the stem tips (3-4 leaf primordia were reserved) of the materials obtained by culture in step 2) were quickly cut with a grafting knife as scions, and the scions were grafted to the incisions of the rootstocks, to make the two be fully attached together to obtain grafted seedlings;

[43] 5) the grafted seedlings obtained in step 4) were cultured in a liquid culture medium (an MS liquid culture medium+40 g/L of sucrose, with pH of 5.8-6.0), a filter paper bridge was used as support during culture, and culture conditions: temperature was 26+2°C, a photoperiod was 16 h/d, and light intensity was 2500 Ix; and

[44] 6) after one week of grafting, the grafted seedlings with dead stem tips were removed in time, further, sprout tillers of the survival grafted seedlings were removed, so as to reduce influence of the sprout tillers on germination of the grafted seedlings, after that, the grafted seedlings were regularly checked, the sprout tillers were removed every other week, when 2 or 3 true leaves grown, the grafted seedlings were transferred to a liquid culture medium (an MT solid culture medium+40 g/L of sucrose) for continuous culture, after 10 d of grafting, a survival rate of the grafted seedlings was observed, after 30 d of grafting, a germination rate of the grafted seedlings was observed, results showed that when grafting was performed by the stab-inoculation method, the survival rate, the germination rate were the highest, when grafting was performed by the cleft grafting method, the survival rate was lowest, the germination 9 rate was 0, and the stab-inoculation method was more conducive to survival and 10103126 germination of grafting (Table 3).

[45] Table 3 Survival rate and germination rate of different grafting methods

Grafting method Survival rate (%) after 10 d of | Germination rate (%) after grafting 30 d of grafting

Cognos [5 fo

Stab-inoculation 82.5 22.5 method

[46] Example 5

[47] According to the operation of Example 4, after about 50 d of grafting, grafted seedlings may grow 4 to 5 true leaves, which are used as transplanting seedlings, specific steps are as follows:

[48] 1) preparation of a transplanting substrate: fine sand, vermiculite and peat soil were mixed according to a mass ratio of 1:1:1, and autoclaved sterilization was performed to obtain sterile culture soil as the transplanting substrate;

[49] 2) before transplanting of the seedlings to be transplanted, the grafted seedlings with stem tips were refined for 3 d under natural scattering light, and then a culture medium at roots of the seedlings to be transplanted was rinsed with tap water;

[50] 3) the transplanting substrate was sub-packaged in a culture bowl, a layer of sterilized bubble beads was spread on a bottom of the bowl, to make culture soil have enough water and air, and the seedlings to be transplanted were planted in the culture bowl, to make the roots naturally disperse in the culture soil, and irrigated with the tap water until a small amount of water flows out from the bottom of the bowl; and the transplanted seedlings were put into seedling grooves in an insect-proof net room, a sunshade net was placed to provide shade and keep ventilation;

[51] 4) in order to keep humidity of the culture bowl about 90%, a small amount of water was regularly sprayed every day for one week after transplantation; an interval between time for spraying the water may be slightly longer after one week, that is, in HU108726 order to prevent the roots of the transplanted seedlings from expanding and eventually rotting due to too much water, the humidity was necessarily appropriately reduced to about 80%; and

[52] 5) after 2-3 weeks, until leaves of the seedlings to be transplanted turned green, and new leaves grown, after the seedlings grown healthily, the sunshade net was put away, and proper sunlight and natural wind were given to the transplanted seedlings; and after 1 to 2 months, when reached 20 cm in height, the transplanted seedlings may be transferred to a large seedling pot and continued to be tended in the isolated net room, to obtain virus-free seedlings of Jinsha pomelo, with an average survival rate of 85%.

[53] According to the above contents, it may be seen that the stem segments having a length of 1-3 cm cut in March to May are disinfected twice with mercuric chloride having a concentration of 0.1 wt.% and sodium hypochlorite having a concentration of 5 wt.%, then the stem tips with 3-4 leaf primordia reserved are grafted with a stab-inoculation method, a survival rate and a germination rate of the grafted seedlings with the stem tips of Jinsha pomelo may be greatly improved, and a pollution rate is reduced.

[54] Although the above examples describe the present invention in detail, the examples are some rather than all of the examples of the present invention. People can also obtain other examples according to the present example without creative efforts, and these examples all fall with the scope of protection of the present invention. 11

Claims

WHAT IS CLAIMED IS: HU108120

1. A virus-free seedling breeding method for Jinsha pomelo, comprising: Picking semi-lignified tender shoots of Jinsha pomelo from March to May, and cutting the tender shoots to 1-3 cm to obtain stem segments of Jinsha pomelo; performing first disinfection on the stem segments of Jinsha pomelo with mercuric chloride having a concentration of 0.1 wt% or sodium hypochlorite having a concentration of 5 wt.%, performing second disinfection with the sodium hypochlorite having a concentration of 5 wt.%, and taking the stem tips with 3-4 leaf primordia reserved as scions; performing the first disinfection and the second disinfection for 10-12 min; grafting the scions onto rootstocks by a stab-inoculation method to obtain grafted seedlings for culture; wherein the stab-inoculation method comprises: under aseptic conditions, removing tops of the rootstocks, to reserve stems having a length of 1.5-2 cm, longitudinally cutting the rootstocks at positions 1 cm away from top ends, horizontally turning the rootstocks by 180 degrees, longitudinally cutting the rootstocks at positions 1 cm away from top ends, connecting the two incisions, cutting through xylem and phloem at one third of stem segments of the rootstocks to form rectangular incisions, and grafting the scions into the rectangular incisions.

2. The breeding method according to claim 1, wherein the stem segments of Jinsha pomelo are provided with 2-3 axillary buds; the stem segments of Jinsha pomelo are stem segments of the semi-lignified tender shoots of Jinsha pomelo with a life cycle within triennial; a disinfection way comprises oscillation and/or shaking; and when disinfected materials and the grafted seedlings are cultured, culture temperature is 26+2‘C, a photoperiod is 16 h/d, and light intensity is 2500 Ix.

3. The breeding method according to claim 2, wherein when the grafted seedlings are cultured, the method comprises: transplanting the grafted seedlings to culture bowls for culture when 4 to 5 true leaves grow. 1

4. Application of the breeding method according to any one of claims 1-3 in cultivating excellent varieties of Jinsha pomelo. 2