A method for breeding a balsa polyploid

Abstract

The application relates to a cultivation method of a lightwood polyploid and relates to the technical field of plant chromosome doubling. The method comprises the following steps: top growth points of lightwood seedlings with a seedling age of 15 days are treated by spraying an inducing liquid to induce chromosome doubling; wherein the inducing liquid comprises: 0.1%-0.4% (w / v) of colchicine, 1 / 4 MS culture medium, 3 mg / L of 6-BA and 0.1% (v / v) of a surfactant Silwet L-77; the spraying treatment is specifically as follows: the top growth points are sprayed with the inducing liquid once every 24 hours, and the spraying is performed for 3 times, and the spraying amount of each time is such that the top growth points are uniformly wetted and liquid drops do not drip. The method can not only effectively improve the lightwood polyploid induction rate, but also control a certain lightwood seedling mortality rate, so that more polyploid lightwood plants can be obtained.

Description

Technical Field

[0001] This application relates to the field of plant chromosome doubling technology, and in particular to a method for cultivating polyploid balsa wood. Background Technology

[0002] Balsa wood (scientific name: Ochroma lagopus Balsa wood, also known as balsa wood, is an evergreen, fast-growing tree belonging to the Malvaceae family and the Balsa genus. It originates from the equatorial region of South America, primarily in Ecuador and Peru. Its timber is widely used in wind power generation and aerospace due to its rapid growth, short growth cycle, low density, uniform texture, and ease of processing. However, my country currently remains highly dependent on imports, and local cultivation faces problems such as mixed varieties, unclear seed sources, excessive branching, and excessive flowering. These undesirable traits lead to high nutrient consumption, severely impacting timber growth and resulting in a low four-year timber maturity rate.

[0003] Polyploids are organisms containing three or more complete sets of chromosomes, and can be divided into autopolyploids and allopolyploids. Their formation pathways mainly include: 1. Diploid genome doubling to form autotetraploids; 2. Hybridization of diploids and tetraploids to form sterile triploids; 3. Interspecific hybridization to form haploid hybrids, which then undergo chromosome doubling to form allopolyploids; 4. Fusion of naturally occurring non-meiotic gametes to form allopolyploids; 5. Hybridization of two autotetraploids to form allotetraploids; 6. Allopolyploids forming new allopolyploids through chromosome recombination.

[0004] Polyploid plants often exhibit significant changes in the morphology of organs such as roots, stems, leaves, flowers, and seeds, including organ enlargement, increased metabolite content, and enhanced stress resistance. Polyploid breeding has already achieved good results in tree species such as poplar and cotton. Therefore, applying this technology to balsa wood holds promise for cultivating new materials with superior agronomic traits.

[0005] Furthermore, soaking or applying colchicine solution (usually water, dilute alcohol, or DMSO) to plant meristems (such as apical and axillary buds) to induce polyploidy is a very classic and widely used method.

[0006] Currently, there are no research reports on polyploid induction in balsa wood in existing technologies. During the applicant's attempt to induce polyploidy in balsa wood using colchicine solutions prepared using traditional methods (usually with pure water or DMSO), the polyploid induction rate was found to be low. Specifically, when different concentrations of colchicine solutions were prepared using pure water (0.1%, 0.2%, and 0.3%), the polyploid induction rate was consistently below 50%, with a maximum of only 47.4% and a corresponding seedling mortality rate of 57.8%. While different concentrations of colchicine solutions were prepared using DMSO (0.1%, 0.2%, 0.3%, and 0.4%), the polyploid induction rate improved somewhat, but still only reached a maximum of 56.3%, with a corresponding seedling mortality rate of 64.5%.

[0007] In the process of inducing polyploidy in balsa wood using colchicine solution, the pursuit of improving the polyploidy induction rate and reducing seedling mortality, thereby obtaining more polyploid plants, has always been the goal of those skilled in the art. Summary of the Invention

[0008] To address or partially address the problems existing in the related technologies, this application provides a method for cultivating polyploid balsa wood, which uses a composite treatment solution to prepare colchicine to induce polyploidy in balsa wood seedlings. This method can not only effectively improve the polyploidy induction rate of balsa wood, but also control a certain mortality rate of balsa wood seedlings, thereby obtaining more polyploid balsa wood plants.

[0009] A method for cultivating polyploid balsa wood seedlings according to this application includes the following steps: spraying an induction solution onto the apical meristem of 15-day-old balsa wood seedlings to induce chromosome doubling; wherein, the induction solution comprises: Colchicine at concentrations of 0.1% to 0.4% (w / v); 1 / 4 MS medium; 3 mg / L of 6-BA; And the surfactant Silwet L-77 at a concentration of 0.1% (v / v); The spraying treatment specifically involves spraying an induction solution onto the apical growth point every 24 hours for a total of 3 sprays. Each spray is applied in such a way that the surface of the apical growth point is evenly moistened without dripping.

[0010] Furthermore, the inducing solution contains colchicine at a concentration of 0.4% (w / v).

[0011] Furthermore, the balsa seedlings are prepared by the following method: fresh seeds obtained by peeling mature balsa capsules harvested no more than 3 days ago are soaked overnight, then treated in a 55°C water bath for 1 hour, and then cultured at 25°C until the seeds break open and show white color. The seeds are then sown in seedling trays to germinate and grow to the corresponding seedling age.

[0012] Furthermore, after spraying the inducing solution, the treatment point is wrapped with moistened absorbent cotton to keep it moist, and the plant is placed in a dark environment for 30-35 days. After that, it is transferred to a strong light environment and the plant is allowed to grow normally by watering and fertilizing.

[0013] This application also provides a method for cultivating triploid balsa wood, comprising the following steps: cultivating 2-year-old tetraploid balsa wood plants using the cultivation method described above; The pollen of the two-year-old tetraploid balsa trees was used as the male parent, and the diploid balsa trees were used as the female parent for hybridization. The hybridization operation is carried out between October of the current year and March of the following year. Specifically, before the flowers of the diploid balsa plant mother plant open, its spiral stamens are removed and the plant is bagged for 1-2 days. Then, the pollen of the tetraploid balsa plant father plant is artificially pollinated onto the stigma of the diploid pistil. After pollination, the ovary develops into a capsule with a length of 10-18 cm. The capsule is harvested after it matures and turns black to obtain triploid balsa seeds.

[0014] Furthermore, during artificial pollination, a solution of 0.1% (v / v) sucrose, 0.05% (v / v) boric acid, and 0.1% (v / v) Silwet L-77 is applied to the stigma of the pistil to improve pollen adhesion.

[0015] Furthermore, the artificial pollination was carried out between 17:30 and 21:00.

[0016] This invention successfully overcomes the technical bottleneck of balancing efficiency and safety in balsa polyploid induction by using an induction solution prepared from 1 / 4 MS medium, 3 mg / L 6-BA, and 0.1% Silwet L-77. Its core mechanism lies in: 1. 1 / 4 MS medium: Provides basic metabolic support and osmotic buffer. MS medium contains a complete range of inorganic salts, vitamins, and organic nutrients. However, this application uses only 1 / 4 MS medium, which provides the nutrients needed to maintain basic cellular metabolism and repair mechanisms while avoiding the excessively high osmotic pressure caused by full-strength medium, which could cause additional damage to seedlings already under colchicine stress.

[0017] 2. 6-BA is a cytokinin that specifically promotes cell division. Colchicine primarily acts on metaphase of mitosis, inhibiting spindle formation. The addition of 6-BA can induce more apical meristem cells to synchronously enter or arrest in the mitotic phase, greatly increasing the number of effective target cells for colchicine. This not only directly increases the absolute probability of chromosome doubling (induction rate), but also, due to more synchronized cell cycles, makes the time window for colchicine's action more defined, thereby reducing the cumulative toxicity caused by prolonged exposure while waiting for all cells to enter the mitotic phase.

[0018] 3. Silwet L-77, as a highly efficient nonionic surfactant, significantly reduces the surface tension of the drug solution. The apical meristem of balsa seedlings is often covered by young leaves or villi, making it difficult for traditional aqueous solutions to adhere evenly and penetrate effectively into the meristematic tissue. Silwet L-77 ensures that the inducing solution is fully wetted, spread, and efficiently penetrates the surface of the meristem, allowing colchicine and 6-BA to reach the target cells more quickly and evenly. This achieves precise and efficient drug delivery: on the one hand, it ensures that sufficient active ingredients reach the site of action, enhancing the induction effect; on the other hand, it avoids excessive toxicity caused by localized drug accumulation or treatment failure due to uneven penetration, thus optimizing safety and efficacy through the route of administration.

[0019] The beneficial effects of this invention are: 1. The induction medium provided in this application, composed of 1 / 4 MS medium, 6-BA, and Silwet L-77, is used for polyploid induction in balsa wood, successfully resolving the technical contradictions of traditional methods where "high induction rate is accompanied by high mortality" or "low mortality but insufficient induction rate." This scheme, through the synergistic effect of its components, achieves a higher polyploid induction rate with an acceptable mortality rate.

[0020] 2. In the scheme described in this application, when colchicine is used at a concentration range of 0.1%-0.4%, the induction rate is consistently maintained at a high level of over 54%, and the mortality rate is controlled below 55%, demonstrating stable performance. This provides a flexible range of concentration options for practical operation.

[0021] 3. The proposed method achieved a peak induction rate of 77.8% at a colchicine concentration of 0.4%, the highest among all experimental groups. Simultaneously, the mortality rate at this point was 54.5%, significantly lower than that of the DMSO matrix at the same induction rate level. Attached Figure Description

[0022] The above and other objects, features and advantages of this application will become more apparent from the more detailed description of exemplary embodiments thereof in conjunction with the accompanying drawings, wherein the same reference numerals generally represent the same components in the exemplary embodiments thereof.

[0023] Figure 1 The balsa seeds and seedlings shown in this application; Figure 2 The chromosome doubling result is determined based on the loss of cells instrument; Figure 3 This is the polyploid flower morphology shown in this application; Figure 4 This application shows a graph of the size and viability of polyploid pollen grains. Detailed Implementation

[0024] The embodiments of this application will now be described in more detail with reference to the examples. While embodiments of this application are shown in the examples, it should be understood that this application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0025] Experimental materials involved in the embodiments of this application Plant materials: Balsa seeds were collected from the 300-mu balsa demonstration base planted by Xishuangbanna Tropical Botanical Garden Technology Co., Ltd.; robust diploid balsa seedlings were germinated in the greenhouse of the central laboratory of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences (21°41' N / 101°25' E, altitude 570 m); treated seedlings were sown at the balsa seedling nursery of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences; and doubled balsa plants were planted in the 7-mu experimental field of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences.

[0026] Other materials: Colchicine (Sigma) alkaloid (Tianjin Xiens Biochemical Technology Co., Ltd.), Alexandrite stain (Macklin), 6-BA, Silwet L-77 and inorganic salts were purchased from CASMA Mall; seedling bags and plastic wrap were purchased from Tianli Supermarket in Menglun Town; substrate nutrient soil was purchased from Chenggong Flower Market in Kunming; medical absorbent cotton and 100 mL spray bottles were purchased from Shanghai Bioengineering Co., Ltd.

[0027] Furthermore, the balsa seedlings used in the examples were all prepared by the following method: 1. Preparation of Balsa seeds Fresh, mature diploid balsa wood naturally pollinated capsules were collected from the balsa wood demonstration forest of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, from April to June. The cottony cilia were manually removed to extract the mature and plump seeds, which were then stored at low temperature in a seed storage cabinet at 4°C.

[0028] 2. Preparation of Balsamic Seedlings: Before germination, select plump, undamaged seeds from the refrigerator and place them in 50 mL centrifuge tubes. Add an appropriate amount of pure water and soak overnight for 12 hours. The next morning, pour out the soaking water, add an appropriate amount of pure water again, and place the tubes in a 55℃ water bath for 1 hour. After treatment, evenly spread the water-absorbed and swollen seeds in a glass petri dish with two layers of filter paper at the bottom. Add 5-10 mL of pure water and soak. Seal the dish with plastic wrap to keep it warm and moist. Transfer the dish to a 25℃ constant temperature incubator for 2-3 days. When the seed coat cracks and the white embryo is exposed, sow the seeds into 8 × 4 seedling trays filled with absorbent and moist substrate. Sow one seed in each seedling hole at a depth of about 1-2 cm. After sowing, cover the trays with plastic wrap to keep them warm and moist. After 7 days, dicotyledonous seedlings will gradually germinate. After 10-30 days, spray the induction treatment solution according to the experimental protocol in the example.

[0029] Figure 1 The image shows balsa seeds and diploid balsa seedlings aged 15 days used in this application.

[0030] In addition, the karyotype of plants treated with colchicine was identified by flow cytometry in this application. Example 1

[0031] Comparison of the effects of different induction solutions on induction rate and mortality. Experimental methods: The apical growth point of diploid balsa seedlings aged 15 days was treated with different induction solutions. The induction solutions are as follows: Pure water; Colchicine solutions with concentrations of 0.1%, 0.2%, and 0.3% were prepared using pure water; Colchicine solutions with concentrations of 0.1%, 0.2%, 0.3%, and 0.4% were prepared using DMSO; Colchicine was dissolved in 1 / 4 MS solution with 3 mg / L 6-BA and 0.1% Silwet L-77 to achieve total solution concentrations of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and 0.6%. The spraying treatment was as follows: The apical growth point was sprayed with an induction solution every 24 hours, for a total of 3 sprays. Each spray was applied until the surface of the growth point was evenly moistened but not dripping (approximately 10 μL of induction solution). After 30 days of recovery culture, the plant survival rate and doubling rate were statistically analyzed, and the results are shown in Table 1 below. It should be noted that the doubling plants counted in this example include not only tetraploid plants (obtained directly from diploid plants by doubling) but also octoploid plants (diploid plants initially showed tetraploid growth, and subsequent treatment with the induction solution resulted in octoploid growth). The results are as follows... Figure 2 As shown, when diploid, tetraploid, and octoploid samples were mixed and tested, three peaks of different sizes appeared.

[0032] Table 1

[0033] The results show: Compared with pure water substrate: At the same colchicine concentration (0.1%-0.3%), the polyploid induction rate of the present invention (54.0%-60.5%) was significantly higher than that of pure water substrate (38.8%-47.4%), with an average increase of about 15 percentage points. Meanwhile, the seedling mortality rate was comparable to or only slightly higher than that of pure water substrate (44.5%-52.3% vs 45.6%-57.8%), indicating that the present invention significantly improved doubling efficiency without significantly increasing toxicity.

[0034] Compared with DMSO substrate: At the same colchicine concentration (0.1%-0.4%), the seedling mortality rate of the method described in this invention (44.5%-54.5%) was significantly lower than that of DMSO substrate (56.7%-71.2%), with an average reduction of approximately 13 percentage points. More importantly, with a lower mortality rate, the induction rate of the method described in this invention (54.0%-77.8%) was comprehensively superior to or equal to that of DMSO substrate (46.2%-56.3%). This demonstrates that the method described in this invention greatly improves the safety of the induction process and simultaneously increases the effective yield. Example 2

[0035] Comparing the effects of different seedling ages of balsa wood on induction rate and mortality rate Experiment: Colchicine was dissolved in 1 / 4 MS solution with 3 mg / L 6-BA and 0.1% Silwet L-77 to a total solution concentration of 0.2%, 0.3%, and 0.4%, respectively. Diploid balsamic seedlings that had germinated for 10, 20, and 30 days were sprayed with the solution. Each group consisted of 30 seedlings, with 3 replicates, for a total of 90 seedlings. The spraying treatment is as follows: spray the apical growth point with an induction solution once every 24 hours, for a total of 3 sprays. Each spray should be applied until the surface of the growth point is evenly moistened but the liquid droplets do not drip (approximately 10 μL of induction solution). After 30 days of recovery culture, the survival rate and induction rate were calculated. The experimental results are shown in Table 2: Table 2

[0036] The results show: Ten-day-old seedlings treated with 0.2% colchicine showed a low mortality rate of 51.11% and a high chromosome doubling efficiency of 47.72%. Seedlings treated with 0.3% and 0.4% colchicine solutions at 20 days of age showed lower mortality rates of 41.11% and 51.11%, respectively, and higher chromosome doubling efficiencies of 35.85% and 43.1%, respectively. Seedlings treated with 0.4% colchicine showed a lower mortality rate of 45.56% and a higher chromosome doubling rate of 33.9% at 30 days old. In summary, the appropriate colchicine concentration varies for seedlings of different ages. The older the seedling, the higher the colchicine concentration. However, when the colchicine concentration exceeds 0.4%, both the mortality rate and chromosome doubling efficiency decrease. Example 3

[0037] Compare the flower morphology, pollen grain size, and activity of polyploids. Flower buds appeared in the experimental field after approximately 1 year and 9 months of transplanting diploid plants and colchicine-treated plants (tetraploid plants). It takes about 60 days from flower bud appearance to flower opening. Diploid and tetraploid flowers were collected between 5 PM and 8 PM on the day of opening and photographed for comparison in the laboratory. 10 mg of pollen was collected, suspended in 10 μL of pure water, and pressed into slides for microscopic observation of size and morphology. The number of pollen grains in different fields of view was counted, and pollen viability was determined using 0.01% Alexander staining solution.

[0038] Further comparison of pollen morphology revealed that tetraploid pollen was larger in size than diploid pollen. Figure 3 The diameter of diploid pollen was 89.05 μm, and the diameter of tetraploid pollen was 107.57 μm, with the difference reaching a highly significant level (Table 4). Pollen viability was compared by staining with Alexandrine stain. Figure 4 The results showed that the viability of diploid pollen was 48.62%, and that of tetraploid pollen was 37.5%. Pollen viability decreased after chromosome doubling (Table 3).

[0039] Table 3 Example 4

[0040] Based on the method for cultivating polyploid balsa wood in Example 1, this example provides a method for cultivating triploid balsa wood seeds, including the following steps: S1. Parental preparation and pollen collection Healthy tetraploid balsa trees with an age of about 2 years were selected as the male parent, and normal diploid balsa trees were selected as the female parent.

[0041] One to two days before the male parent plant's flowers open, the flower spikes are isolated by bagging. When the calyx is about to split open, revealing the white petals (the "white-revealing stage"), the bag is removed, and the flowers that are about to open are collected. By peeling off the calyx, the pollen is collected with a soft brush into a sterile container and placed in a cool, dark place for later use. This method ensures the acquisition of pure, highly viable pollen.

[0042] S2. Treatment of the parent plant and artificial pollination Before the flower buds of the mother plant (diploid) swell but the flowers open, carefully remove all the spiral stamens with tweezers, and then immediately cover the plant with a paper bag to isolate it and prevent accidental pollination.

[0043] One to two days after bagging, when the stigma of the pistil has matured and secretes mucus, pollination can be carried out. Pollination should be performed between 5:30 PM and 9:00 PM. Before pollination, preferably, a mixed solution containing 0.1% (v / v) sucrose, 0.05% (v / v) boric acid, and 0.1% (v / v) Silwet L-77 should be evenly applied to the stigma of the female parent with a cotton swab to enhance pollen adhesion.

[0044] Then, using a pollination tool (such as a brush or cotton swab), take the tetraploid pollen prepared in step S1 and gently and evenly apply it to the stigma of the maternal parent.

[0045] S3. Post-pollination management and seed harvesting After pollination, re-cover the plant with a paper bag and label it. Remove the paper bag after about one week. The ovary develops normally, forming a capsule 10-18 cm in length after about 4 months of growth. Harvest the capsule when it is fully mature and the pericarp turns dark brown. Extract the seeds from the capsule; these are the target triploid balsa seeds.

[0046] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for cultivating polyploid balsa wood, characterized in that, Includes the following steps: The apical meristem of 15-day-old balsa seedlings was treated with an induction solution to induce chromosome doubling; wherein the induction solution consisted of the following components: Colchicine at concentrations of 0.1% to 0.4% (w / v); 1 / 4 MS medium; 3 mg / L of 6-BA; And the surfactant Silwet L-77 at a concentration of 0.1% (v / v); The spraying treatment specifically involves spraying an induction solution onto the apical growth point every 24 hours for a total of 3 sprays. Each spray is applied in such a way that the surface of the apical growth point is evenly moistened without dripping.

2. The method for cultivating polyploid balsa wood according to claim 1, characterized in that: The concentration of colchicine in the induction solution was 0.4% (w / v).

3. The method for cultivating polyploid balsa wood according to claim 2, characterized in that: The balsa seedlings are prepared by the following method: fresh seeds obtained by peeling mature balsa capsules harvested no more than 3 days ago are soaked overnight, then treated in a 55°C water bath for 1 hour, and then cultured at 25°C until the seeds break open and show white color. The seeds are then sown in seedling trays to germinate and grow to the corresponding seedling age.

4. The method for cultivating polyploid balsa wood according to claim 1, characterized in that: After spraying the inducing solution, wrap the apical growth point with moistened absorbent cotton to keep it moist, and place the plant in a dark environment for 30-35 days. After that, transfer it to a strong light environment and allow the plant to grow normally by watering and fertilizing.

5. A method for cultivating triploid balsa wood, characterized in that, Includes the following steps: Two-year-old tetraploid balsa trees were obtained by cultivating the cultivation method according to any one of claims 1-4; The pollen of the two-year-old tetraploid balsa trees was used as the male parent, and the diploid balsa trees were used as the female parent for hybridization. The hybridization operation is carried out between October of the current year and March of the following year. Specifically, before the flowers of the diploid balsa plant mother plant open, its spiral stamens are removed and the plant is bagged for 1-2 days. Then, the pollen of the tetraploid balsa plant father plant is artificially pollinated onto the stigma of the diploid pistil. After pollination, the ovary develops into a capsule with a length of 10-18 cm. The capsule is harvested after it matures and turns black to obtain triploid balsa seeds.

6. The method for cultivating triploid balsa wood according to claim 5, characterized in that: The artificial pollination was carried out between 5:30 p.m. and 9:00 p.m.

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