CN114303945B

CN114303945B - In-vitro preparation method of Chinese rose fertile pollen

Info

Publication number: CN114303945B
Application number: CN202111302309.2A
Authority: CN
Inventors: 徐子岩; 朱木兰; 周慧晶; 郑珂瑗
Original assignee: SHANGHAI CHENSHAN BOTANICAL GARDEN
Current assignee: SHANGHAI CHENSHAN BOTANICAL GARDEN
Priority date: 2021-11-04
Filing date: 2021-11-04
Publication date: 2023-03-17
Anticipated expiration: 2041-11-04
Also published as: CN114303945A

Abstract

The method takes semi-lignified Chinese rose branches as explants, induces the Chinese rose bottle to bloom through the steps of high-frequency synchronous adventitious bud induction, adventitious bud elongation, chinese rose reproductive conversion, fertile pollen induction culture and the like, generates functional pollen, and establishes the in vitro preparation method of the Chinese rose fertile pollen. The method can meet the growth conditions of the Chinese rose in nature so that the Chinese rose is not subjected to the growth of space-time factors, provides materials for test tube hybridization and accelerates the breeding period; meanwhile, the generated fertile pollen can be used as a starting material for haploid culture, and an excellent material is provided for breeding of Chinese roses.

Description

In-vitro preparation method of Chinese rose fertile pollen

Technical Field

The invention belongs to the field of plant tissue culture and breeding, and particularly relates to an in-vitro preparation method of rose fertile pollen.

Background

China rose (Rosa chinensis Jacq.), a plant of Rosa (Rosaceae Juss.), called 'queen in flower', one of ten big flowers in China, one of the four big cut flowers in the world (China rose development report (2 nd edition) [ J ]. Agricultural science and technology and information (modern garden), 2014,11 (05): 44-47+ 1-43.), has very high ornamental value and economic value (Ma Xuefan. Chinese rose cross breeding research [ J ]. Farmer colludes, 2019 (17): 18.). With the development of times, the breeding direction of the roses mainly focuses on the appearance and ornamental characteristics of varieties, such as color, plant type, flower fragrance and the like (Hu Qingpo, li Xuefeng, wu Qiong, li Xinwei, meng Yuanyuan, liu Wei. Modern roses breeding research [ J ]. Beijing agriculture, 2014 (24): 98-99.). Professor Gudin in France develops the research work of culture of Chinese rose embryos by exploring fertility of pollen, incompatibility of pollen and stigma, and the like under various conditions. Chinese scholars have achieved stage performance in the aspects of Chinese rose resource collection, protection, breeding and the like, and a plurality of new varieties of outdoor cultivation are bred for arid and cold areas. However, the growth of different Chinese roses is limited by time-space conditions such as seasons, climates and the like, the breeding period is long, the in-bottle flowering technology can accelerate the breeding process, the flowering of the Chinese roses can be artificially promoted through 1-2 culture periods, individuals with excellent characters are purposefully bred according to the flowering condition, the workload of cultivating invisible flower varieties in large quantities is reduced, and the breeding work is more pertinent (Wu Gaoqiong. SSR molecular marker-based wild parent analysis of the Chinese ancient Chinese roses [ D ]. Yunnan university, 2019.).

In the process of hybridization breeding of modern China roses, most of China roses are high hybrids, and the hybridization breeding work among varieties faces great difficulty (Ma Xuefan. China rose hybridization breeding research [ J ]. Farmer consummation, 2019 (17): 18.), haploid breeding can enable the hybrids to become homozygous, so that pollen becomes one of key factors for breeding new varieties (Li Zedi, du Ying, jiang Xueru, lu Yuexiu, ren Ruifen, zhang Yixuan, liuyan, 6 China roses ultralow temperature pollen preservation research [ J ]. Southwest agriculture news, 2019,32 (06): 1376-1382.). In nature, the generation of pollen is limited by seasons, and parents have the problems of flowering asynchronism, short pollen life and the like (Shang Xiaoqian. Peony pollen cryopreservation research [ D ]. Beijing forestry university, 2005 ]), so that the application of the pollen in haploid breeding, crossbreeding and the like is seriously influenced. Cryopreservation is a new technology developed in recent decades and is considered as the only way for long-term storage of pollen, but the problems of complex operation, low pollen activity and the like exist (Li Zedi. Evaluation of the ultralow-temperature long-term storage effect of garden plant pollen and research on physiological mechanism thereof [ D ]. Beijing university of forestry, 2019.), and breeding materials can be obtained throughout the year when test tube flowering of Chinese roses breaks through natural condition limitation, physiological isolation and the influence of parent-parent flowering.

Disclosure of Invention

The method takes strong semi-lignified Chinese rose branches growing in spring and autumn as explants, induces the Chinese rose bottle to bloom through the steps of high-frequency synchronous adventitious bud induction, adventitious bud elongation, chinese rose reproductive conversion, fertile pollen induction culture and the like, generates functional pollen, establishes a Chinese rose pollen in vitro induction system, meets the growth conditions of the Chinese rose in nature so that the Chinese rose is not subjected to the growth of space-time factors, provides materials for test tube hybridization, and accelerates the breeding period; meanwhile, the generated fertile pollen can be used as a starting material for haploid culture, and an excellent material is provided for breeding of Chinese roses.

The technical scheme provided by the invention is as follows: an in vitro preparation method of regenerated or fertile Chinese rose pollen, which comprises the following steps:

1) Preparation of explant material: taking robust semi-lignified Chinese rose branches growing in spring or autumn, and disinfecting the branches for later use;

2) High-frequency synchronous adventitious bud induction:

(a) Inoculating the sterilized explant into MS +2.5-3.5 mg/L6-BA +0.25-0.35mg/L NAA +0.1-0.3% PPM (Plant regenerative mix, PPM) culture medium until sprout grows out;

(b) Cutting off the top of the bud seedling in the step (a), reserving a base part, transferring the bud seedling to a culture medium of MS +1.5-3 mg/L6-BA +0.15-0.3mg/L NAA, and inducing an adventitious bud cluster with a certain synchronous effect;

(c) Selecting the adventitious bud cluster with high synchronization rate in the step (b) to be transferred to a culture medium of MS +0.5-2 mg/L6-BA +0.05-0.2mg/L NAA +0.1-0.5mg/L KT;

3) Adventitious bud elongation: inoculating the adventitious bud induced in the step 2) into an adventitious bud elongation induction culture medium, wherein the basic culture medium is an MS culture medium, and 0.1-0.5 mg/L6-BA and 0.01-0.05mg/L NAA are added;

4) And (3) reproductive transformation: cutting bud seedling with height above 3cm, inoculating to 2/3MS +0.5-1.5 mg/L6-BA +0.05-0.15mg/L NAA +0.3-0.7mg/L ABA +0.05-0.15g/L KH ₂ PO ₄ Culturing on a culture medium until buds appear; thus, the regeneration of the Chinese rose is realized.

Further, the method also comprises the following steps:

5) And (3) induction of fertile pollen: transferring the plants with the flower buds into MS +0.5-1.5 mg/L6-BA +0.05-0.15mg/L NAA +3-5mg/L H BO3+35-45g/L sucrose culture medium for culturing until the plants bloom.

More specifically, the invention provides a method for preparing Chinese rose fertile pollen in vitro, which comprises the following steps:

1. preparation of explant material: taking strong semi-lignified Chinese rose branches in spring or autumn, washing the branches with running water for 0.5h, shaking and washing 5 per thousand potassium permanganate for 5min, placing the branches on a superclean workbench, treating with 75% ethanol for 30s, soaking with 50% plant tissue culture antibacterial protective agent for 8-15min, washing with sterile water for 3-5 times, and inoculating the branches on an MS culture medium added with 3 mg/L6-BA, 0.3mg/L NAA and 0.1-0.5 PPM (V/V). The PPM treatment time is preferably 10min, the disinfection effect is best, the cleaning rate is 95.33%, the survival rate is 97.9%, and the explant activity is strong.

2. High frequency synchronous adventitious bud induction

(1) The sterilized explants were inoculated into MS +3 mg/L6-BA +0.3mg/L NAA +0.2% PPM medium and cultured for 2 weeks.

(2) Cutting off the top end of the extended bud seedling in the step (1) to reserve a base part, transferring the base part to a culture medium of MS +1.5-3 mg/L6-BA +0.15-0.3mg/L NAA, and culturing for 4 weeks to induce an adventitious bud cluster with a certain synchronous effect. Preferably, 2mg/L of 6-BA and 0.2mg/L of NAA are added, so that the induction effect of the adventitious bud is best, the induction rate reaches 93.33 percent, and the average bud number is 7.33.

(3) And (3) selecting the adventitious bud cluster with higher synchronization rate in the step (2) to be transferred to a culture medium of MS +1-2 mg/L6-BA +0.1-0.2mg/L NAA +0.1-0.5mg/L KT, and culturing for 4 weeks to obtain a large number of adventitious bud clusters with synchronous growth and development. The high-frequency synchronous induction effect of MS +1 mg/L6-BA +0.1mg/L NAA +0.5mg/L KT is the best, the induction rate is the highest and reaches 87.41%, and the average bud number is 13.94.

3. Adventitious bud elongation

(4) And (4) inoculating the adventitious buds induced in the step (3) into an adventitious bud elongation induction culture medium, wherein the basic culture medium is an MS culture medium, 0.1-0.5mg/L of 6-BA and 0.01-0.05mg/L of NAA are added, the culture is carried out for 4 weeks, the bud elongation effect and the growth condition of the culture medium added with 0.3mg/L of 6-BA and 0.03mg/L of NAA are optimal, the adventitious bud elongation rate reaches 78.65%, and the average bud length is 47.80mm.

4. Conversion of Chinese rose into reproductive system

(5) Cutting bud seedlings with the height of more than 3cm in the step (4) and inoculating the bud seedlings to 2/3MS + 1mg/L6-BA +0.1mg/L NAA +0.3-0.5mg/L ABA +0.05-0.1g/L KH ₂ PO ₄ Cultured on the medium for 4 weeks. 0.5mg/L ABA and 0.1g/LKH were added ₂ PO ₄ The induction effect is optimal, and the bud rate is 54.44%.

5. Fertile pollen induction

(5) Transferring the bud plant in the step (5) to MS +1 mg/L6-BA +0.1mg/L NAA +3-5mg/L H ₃ BO ₃ Culturing in +40g/L sucrose culture medium, preferably 5mg/L H ₃ BO ₃ The flower bud induction rate is 53.33%, the flowering rate is 68.25%, the pollen blooms after 2-3 weeks of culture, and the vitality of the pollen prepared in vitro has no significant difference from the vitality of the pollen formed under the natural condition. The electron microscope result also shows that the isolated prepared pollen has no difference from naturally formed fertile pollen in the characteristics of the size, shape, ornamentation and the like.

6. Culture conditions

All the culture media are added with 30g/L of sucrose, 0.1g/L of inositol and 5g/L of agar powder except that the sucrose concentration in the step (5) is changed, and the pH value of the culture medium is adjusted to be 5.8. The culture conditions are 25 + -2 deg.C, illumination intensity is 2000-3000lx, and illumination is 16h/d.

According to the invention, through experimental research and analysis of Chinese rose characteristics, the establishment of a Chinese rose pollen in-vitro induction system is finally completed, wherein different technical measures, particularly the composition and operation treatment (more particularly the treatment of reproductive transformation) of a culture medium are adopted in different link researches, and effective fertile pollen is generated through electron microscope observation. The invention has strong practicability, can provide materials for test tube hybridization by only finishing the induction of the pollen in the culture bottle, quickens the breeding period, and the generated fertile pollen can be used as the initial material for haploid culture and provides excellent materials for the breeding of Chinese roses.

Drawings

FIG. 1 high frequency synchronous adventitious bud induction of rose. Wherein, A cultures explants for 2 weeks; b, adventitious bud clusters with certain synchronous effect; c high frequency synchronous adventitious bud clump.

FIG. 2 elongated adventitious bud of rose.

Fig. 3 rose reproductive transition. Wherein, the left rose is converted into initial bud seedlings by reproduction; the right picture shows the bud of Chinese rose.

Fig. 4 flowering of Chinese rose. Wherein, the bud emergence day 15 of the A Chinese rose; and B, flowering of Chinese rose.

FIG. 5 is an SEM photograph of pollen. Wherein, A is the equatorial plane view (5000 x) of the pollen under natural conditions; b equatorial plane view (5000 x) of the isolated induced pollen; c, polar view of pollen under natural conditions (6000 x); d, performing isolated culture on pollen to obtain a polar view (6000 x); e, natural condition pattern of pollen (20000 x); f, the ornamentation of the pollen is cultured in vitro (20000 x).

FIG. 6 shows that the China rose of 'hometown thinking' variety after reproductive transformation blooms.

Detailed Description

The following detailed description of specific embodiments of the invention is, however, not to be taken in a limiting sense.

1. Preparation of explant Material

Washing strong monthly rose branches of semi-lignified celestial mirror variety in spring for 0.5h in running water, shaking and washing for 5min by 5 per thousand potassium permanganate, placing on an ultra-clean workbench, treating with 75% ethanol for 30s, soaking in 50% Plant tissue culture antibacterial protective agent (Plant advanced Mixture, PPM) for 5-15min (table 1), and inoculating to MS culture medium supplemented with 3 mg/L6-BA, 0.3mg/L NAA, and 0.1-0.5% PPM (V/V). After 2 weeks of culture, the contamination and growth of each treated material were counted. The result shows that the cleaning rate is gradually increased along with the prolongation of the PPM treatment time, the cleaning rate of the PPM treatment for 15min reaches 98 percent, and the survival rate is gradually reduced from 100 percent to 84.35 percent. Excessive PPM treatment can cause partial explant inactivation or growth inactivation, and the optimal disinfection treatment is PPM 10min, the cleanliness rate is 95.33%, and the survival rate is 97.9%.

TABLE 1 Effect of the Sterilization method on the Sterilization Effect of starting materials

Note: each treatment was inoculated with 50 explants and repeated 3 times.

2. High frequency synchronous adventitious bud induction

(1) Inoculating the sterilized explant into MS +3 mg/L6-BA +0.3mg/L NAA +0.2% PPM medium, and culturing for 2 weeks to obtain elongated sprout (FIG. 1-A).

(2) Cutting off the top of the extended bud seedling in the step (1) to reserve a base part, transferring the base part to a culture medium of MS +0.5-3mg/L mg/L6-BA +0.05-0.3mg/L NAA, and culturing for 4 weeks to induce an adventitious bud cluster with a certain synchronous effect (figure 1-B). The statistical results show (Table 2), the adventitious bud induction rate and the average adventitious bud number both show a trend of increasing first and then decreasing with the increase of the 6-BA and NAA concentrations. Wherein, when the combination of the growth regulator is 2 mg/L6-BA +0.2mg/L NAA, the induction effect of the adventitious bud is best, the induction rate reaches 93.33 percent, and the average bud number is 7.33.

(3) And (3) selecting the adventitious bud cluster with higher synchronization rate in the step (2) to be transferred to a culture medium of MS +1-2 mg/L6-BA +0.1-0.2mg/L NAA +0.1-0.5mg/L KT, and culturing for 4 weeks to obtain a large number of adventitious bud clusters with synchronous growth and development (figure 1-C). Counting the number of effective buds with the height of more than 1cm and the high-frequency synchronization rate. The result is shown in Table 3, the high-frequency synchronous growth of the China rose is facilitated by the KT with higher concentration, wherein the high-frequency synchronous induction effect of MS +1 mg/L6-BA +0.1mg/L NAA +0.5mg/L KT is the best, the induction rate is the highest and reaches 87.41%, and the average bud number is 13.94.

TABLE 2 Effect of different phytohormone combinations on preliminary synchronization of adventitious buds

TABLE 3 Effect of different phytohormone combinations on high frequency synchronized adventitious bud Induction

3. Elongation of adventitious bud

(4) Inoculating the adventitious bud induced in the step (3) into an adventitious bud elongation induction culture medium, wherein the basic culture medium is an MS culture medium, adding 0.1-0.5 mg/L6-BA and 0.01-0.05mg/L NAA, culturing for 4 weeks, and counting the height and elongation of the adventitious bud. The results showed that adventitious buds were elongated by each treatment, but the effect of adventitious bud elongation was greatly different depending on the concentration of the growth regulator used in the treatment (Table 4). With the increase of the growth regulator, the elongation rate of the adventitious bud and the average seedling height tend to increase firstly and then decrease, the elongation effect and the growth condition of the bud seedling treated by 0.3 mg/L6-BA and 0.03mg/L NAA are optimal (figure 2), when the bud seedling is cultured for 4 weeks, the elongation rate of the adventitious bud reaches 78.65%, the average bud seedling length is 47.80mm, and the growth state is good.

TABLE 4 Effect of different hormone combinations on adventitious bud elongation of Rosa chinensis

4. Reproductive transformation

(5) Cutting bud seedling (figure 3-A) with height more than 3cm in the step (4) and inoculating the bud seedling to 2/3MS + 1mg/L6-BA +0.1mg/L NAA +0-7mg/L ABA +0-0.2g/L KH ₂ PO ₄ Cultured on the medium for 4 weeks. Each treatment was inoculated with 10 flasks, 1 strain per flask, and repeated 3 times. And counting the number of buds and the bud rate. The results show (Table 5) that proper amounts of ABA and KH were added to the medium ₂ PO ₄ Is beneficial to the reproductive conversion of Chinese roses, the number of flower buds and the bud emergence rate show the trend of rising firstly and then falling along with the increase of the treatment concentration, 0.5mg/L ABA and 0.1g/LKH are added ₂ PO ₄ The induction effect is best (figure 3), and the bud ratio is 54.44%.

TABLE 5 different concentrations of ABA and KH ₂ PO ₄ Effect of combinations on the reproductive transformation of Rosa chinensis

5. Fertile pollen induction

(6) Transferring the bud plant in the step (5) to MS +1 mg/L6-BA +0.1mg/L NAA +0-7mg/L H ₃ BO ₃ +40g/L sucrose culture medium, culturing for 2-3 weeks, observing and counting the flowering rate and days required for flowering of each treatment. By means of I ₂ KI staining method for detecting the viability of rose pollen (CK) formed under natural conditions and rose pollen induced in the bottle. Observing and recording the pollen formed under natural conditions and the pollen shape, equatorial plane view, polar plane view, ornamentation and other features induced in the bottle by an electron microscope method. The results show (Table 6) that addition of a suitable amount of boric acid to the medium is beneficial to the reproductive transformation of roses and the induction of viable pollen with H ₃ BO ₃ The concentration is increased, the flowering rate is in the trend of ascending first and descending second, the days required for flowering are reduced first and then increased, and 5mg/L H is added ₃ BO ₃ The induction effect is the best (figure 4), the flowering rate is 68.25%, the pollen is cultivated for about 30 days to bloom, and the vigor of the pollen prepared in vitro has no significant difference from the vigor of the pollen formed under the natural condition. The electron microscope results also show that the pollen generated by tissue culture induction is not different from naturally-formed fertile pollen in the characteristics of size, shape, texture and the like (figure 5).

TABLE 6 Effect of different boric acid concentrations on the induction of fertile pollen of roses

Note: each treatment was inoculated with 10 explants and repeated 3 times.

Meanwhile, the present inventors also induced pollen in the Hovenia variety under the above-identified optimal conditions, and shown in FIG. 6, the induced rose blossoms.

Claims

1. A method for regenerating Chinese rose and preparing fertile pollen in vitro comprises the following steps:

2) High frequency synchronous adventitious bud induction

(a) Inoculating the sterilized explant into MS +2.5-3.5 mg/L6-BA +0.25-0.35mg/L NAA +0.1-0.3% PPM +30 g/L sucrose +0.1 g/L inositol + 5g/L agar powder, and adjusting the pH value to 5.8 to grow the sprout;

(b) Cutting off the top reserved base of the sprout in the step (a), transferring to MS +1.5-3 mg/L6-BA +0.15-0.3mg/L NAA +30 g/L sucrose +0.1 g/L inositol + 5g/L agar powder, adjusting the pH value to be 5.8 culture medium, and inducing an adventitious bud cluster with a certain synchronization effect;

(c) Selecting the adventitious bud cluster with high synchronization rate in the step (b) to be transferred to MS +0.5-2 mg/L6-BA +0.05-0.2mg/L NAA +0.1-0.5mg/L KT +30 g/L cane sugar +0.1 g/L inositol + 5g/L agar powder, and adjusting the pH value to be 5.8 of the culture medium;

3) Elongation of adventitious bud

Inoculating the adventitious buds induced by the step 2) into a MS culture medium +0.1-0.5 mg/L6-BA +0.01-0.05 mg/L NAA +30 g/L cane sugar +0.1 g/L inositol + 5g/L agar powder, and adjusting the pH value to be 5.8;

4) Reproductive transformation

Cutting sprout with height above 3cm, inoculating to 2/3MS +0.5-1.5 mg/L6-BA +0.05-0.15mg/L NAA +0.3-0.7mg/L ABA +0.05-0.15g/L KH ₂ PO ₄ +30 g/L sucrose +0.1 g/L inositol + 5g/L agar powder, and culturing on a culture medium with the pH value adjusted to 5.8 until buds appear, so as to realize the regeneration of the Chinese rose;

further comprising the steps of: 5) Fertile pollen induction

Transferring the plants with the flower buds to MS +0.5-1.5 mg/L6-BA +0.05-0.15mg/L NAA +3-5mg/L H ₃ BO ₃ +35-45g/L sucrose medium until flowering.

2. The method of claim 1, wherein: the sterilization operation of the step 1) is as follows: washing with potassium permanganate, washing with ethanol, soaking in plant tissue culture antibacterial protective agent, and washing with sterile water.

3. The method of claim 2, wherein: the sterilization operation of the step 1) is as follows: flushing with flowing water for 0.5h, shaking and washing with 5 ‰ potassium permanganate for 5min, placing on a superclean bench, treating with 75% ethanol for 30s, soaking with 50% plant tissue culture antibacterial protective agent for 8-15min, and washing with sterile water for 3-5 times.

4. The method of claim 1, wherein: in the step 2) (a), the culture medium is MS +3 mg/L6-BA +0.3mg/L NAA +0.2% PPM +30 g/L cane sugar +0.1 g/L inositol + 5g/L agar powder, the pH value is adjusted to 5.8, and the culture time is 10-20 days; in the step 2) (b), the concentration of 6-BA in the culture medium is 2mg/L, NAA and the concentration of 6-BA in the culture medium is 0.2mg/L, and the culture time is 25-35 days.

5. The method of claim 4, wherein: in the step 2) (c), the culture medium is MS +1 mg/L6-BA +0.1mg/L NAA +0.5mg/L KT +30 g/L cane sugar +0.1 g/L inositol + 5g/L agar powder, the pH value is adjusted to be 5.8, and the culture time is 25-35 days.

6. The method of claim 1, wherein: the concentration of 6-BA in the culture medium of the step 3) is 0.3mg/L, NAA, the concentration of 6-BA in the culture medium is 0.03mg/L, and the culture time is 25-35 days.

7. The method of claim 1, wherein: in the step 4), the culture medium is 2/3MS +1 mg/L6-BA +0.1mg/L NAA +0.5mg/L ABA+0.1 g/L KH ₂ PO ₄ +30 g/L sucrose +0.1 g/L inositol + 5g/L agar powder, pH adjusted to 5.8.

8. The method of claim 7, wherein: the culture time in step 4) is 25 to 35 days.

9. The method of claim 1, wherein: the culture medium in the step 5) is MS added with 6-BA with the concentration of 1mg/L, the concentration of NAA is 0.1mg/L, and H ₃ BO ₃ The concentration of (2) is 4 mg/L sucrose concentration is 40g/L and cultivation is carried out until flowering.

10. The method of any of claims 1 to 7, wherein: the culture conditions of each step are 25 +/-2 ℃, the illumination intensity is 2000-3000lx, and the illumination is 16h/d.