阅读说明：本技术 一种利用2n花粉杂交选育荷花三倍体的方法 (Method for breeding lotus triploid by 2n pollen hybridization ) 是由 刘凤栾 田代科 秦密 张大生 刘青青 付乃峰 于 2021-01-06 设计创作，主要内容包括：一种利用2n花粉杂交选育荷花三倍体的方法,诱导荷花雄蕊稳定高效地产生2n花粉,再与二倍体植株有性杂交得到三倍体莲子,萌发和栽培后获得三倍体植株；诱导时,选择生长健壮、长度为3～3.5cm的荷花花蕾,拨开花蕾外层花瓣,内侧留一层花瓣包裹雄蕊群,在拨开的外层花瓣与保留的内层花瓣之间填充棉质物,两次滴加浓度为0.02～0.03wt.％的秋水仙素溶液,雄蕊成熟散粉后进行人工授粉,获得成熟三倍体莲子。处理方式稳定,诱导产生2n花粉的成功率高,可大批量地处理花蕾,展开定向杂交与自交,选育过程简便,育种周期短,获得的三倍体株型大,花量增加,显花度高,花朵颜色鲜艳,增加了荷花三倍体的多样性。(A method for breeding lotus triploid by 2n pollen hybridization comprises inducing lotus stamen to stably and efficiently generate 2n pollen, sexual hybridizing with diploid plant to obtain triploid lotus seed, germinating and cultivating to obtain triploid plant; during induction, a lotus bud which is robust in growth and 3-3.5 cm long is selected, the outer petals of the bud are poked, a layer of petals is left on the inner side of the bud to wrap a stamen group, cotton is filled between the poked outer petals and the reserved inner petals, colchicine solution with the concentration of 0.02-0.03 wt.% is dripped twice, and artificial pollination is carried out after the stamens are mature and loose powder, so that the mature triploid lotus seed is obtained. The treatment mode is stable, the success rate of inducing to generate 2n pollen is high, the flower buds can be treated in large batch, directional hybridization and selfing can be developed, the breeding process is simple and convenient, the breeding period is short, the obtained triploid plants are large in size, the flower quantity is increased, the flower development degree is high, the flower color is bright, and the variety of the lotus triploid is increased.)

1. A method for breeding lotus triploid by 2n pollen hybridization comprises the following steps:

1) induction of 2n pollen

Selecting a lotus flower bud which is robust in growth, 3-3.5 cm in length and milky-white in stamen group, poking outer petals of the flower bud in the morning, only reserving 4-6 petals of the innermost layer to wrap the stamen group, filling cotton matter between the poked outer petals and the reserved inner petals, wherein the filling thickness is 3-4 mm, dropwise adding 2-3 ml of colchicine solution with the concentration of 0.02-0.03 wt.% into the cotton matter to enable the cotton matter to be saturated with water, resetting the poked outer petals and inducing;

after inducing for 20-30 hours, dripping colchicine solution with the concentration of 0.02-0.03 wt.% again until the cotton is saturated with water, inducing for 20-30 hours, removing the cotton, and taking stamens of the cotton after the flowers bloom;

2) artificial pollination for hybridization

Collecting the stamen group of loose powder in the step 1) as a male parent, selecting a lotus flower on the day 2 of blooming as a female parent, sleeving a tuck net on the female parent before blooming, removing the male parent, clamping the loose powder stamens, smearing the pistil stigma of the female parent before 10 am, and sleeving the tuck net back after finishing;

and (5) after pollination for 25-30 days, the lotus seedpod grows mature, the lotus seeds are brown or black brown, and the lotus seeds are peeled off.

2. The method for breeding the lotus triploid through 2n pollen hybridization according to claim 1, wherein in the step 1), after removing the cotton for 8-10 days, the treated bud grows mature and is about to bloom, the bud is covered with a string bag, and after 2-3 days, the petals of the mature bud are gradually opened, 4-7 points in the morning of the 2 nd day of blooming, anthers of pistil groups are cracked, and pollen is scattered.

3. The method for breeding lotus triploid by 2n pollen hybridization according to claim 1, wherein in step 1), the cotton substance is absorbent cotton, cotton or cotton yarn.

4. The method for breeding the lotus triploid through 2n pollen hybridization according to claim 1, wherein in the step 1), each induction time is 20-25 hours.

5. The method for breeding lotus triploid by 2n pollen hybridization as claimed in claim 1, wherein the time for inducing the flower bud in step 1) is 7-10 am.

6. The method for breeding lotus triploid by 2n pollen hybridization as claimed in claim 1, wherein in step 1), the flower bud is covered with paper bag after the opened outer petals are reset.

7. The method for breeding lotus triploid with 2n pollen hybridization as claimed in claim 1, wherein in step 2), the pollen-dispersed stamens are collected between 7-8 o' clock earlier on the 2 nd day of flower blooming of the treated bud when the male parent is collected.

8. The method for breeding lotus triploid by 2n pollen hybridization according to any of the claims 1-7, wherein the flower bud is american lotus bud.

Technical Field

The invention belongs to the field of lotus breeding, and particularly relates to a method for breeding lotus triploid by 2n pollen hybridization.

Background

The lotus is a lotus (Nelumbo Adans.), is one of ten traditional famous flowers in China, is also a national flower in India and Vietnam and a world famous religious holy flower, and has extremely high cultural connotation and ornamental value. Meanwhile, lotus roots are one of important vegetables in China; lotus seed, flower and leaf can be used as the medicine; can also be used for purifying and restoring ecological environment; therefore, the lotus also has very important economic value.

To date, there are only two types of lotus, asian lotus distributed in northern asia and australia, and american lotus located in north america and central america. The Asian lotus has various cultivars, including single-petal, double-petal, thousand-petal and other flower types, and the flower color is red to pink or white; most of the American lotus is still in a semi-wild or natural state, and is only in faint yellow, so that the variety is less. The two are geographically isolated but cross-fertile with each other.

At present, the names of lotus varieties recorded in official documents in the world reach 2080, China is the country with the most recorded varieties, and then Japan and the United states are the second place, while Europe and Africa are few. In the 21 st century, techniques such as artificial physical ray radiation breeding, chemical mutation breeding and the like are introduced into improved lotus seed breeding, but the traditional sexual cross breeding is still the main method.

Although the breeding work of the lotus is paid more and more attention by countries and breeders, and a plurality of excellent varieties exist, compared with Chinese roses, chrysanthemums and even water lily, the abundance of the lotus in various aspects such as flower types, flower colors, resistance and the like is low, and the market competitiveness is difficult to promote.

Moreover, no matter the lotus seeds are used for cultivating, viewing and restraining landscapes and courtyards or for eating lotus roots and lotus seeds, the market demand of improved lotus seeds is extremely large, the practical cultivation is only limited on a few traditional varieties, and the varieties with high viewing performance and strong stress resistance are few. Therefore, the development of multi-level, multi-path and multi-directional fine variety breeding work is one of the important means for improving the development of the lotus industry.

In the prior art, the breeding research on the polyploid of the lotus is less, and the lotus seed or the lotus root stem tip is treated by colchicine in the conventional doubling method; or the whole flower bud is directly wrapped by absorbent cotton for induction treatment or liquid is directly injected into the flower bud, the former has poor induction effect on lotus varieties with extremely heavy petals or thick petals, and the latter liquid directly soaks pistil and stamen groups, so that the gynoecia and the androecia are often poor in development, and a large amount of viable 2n pollen cannot be obtained.

Huang Guo Zheng et al (method for artificially inducing polyploid with lotus, journal of horticulture, 2(10):143-144, 1983) performed colchicine solution soaking and bud dropping treatment on germinated plumule of lotus seed, and doubled plants were obtained by the soaking treatment method, while doubled plants were not obtained by the bud dropping treatment.

The method comprises the following steps of (duro \32675) (preliminary research on growth characteristics and certain economic character performances of the lotus triploid, journal of plant science, 3 (002): 119-. The premise for the triploid culture in this method is that tetraploid parents are required, and it is not mentioned how to obtain tetraploid parents.

The seed selection of tetraploid parents generally needs to induce seeds or bud points to screen, at least 2-3 times or years are needed for seedling formation, identification and stability detection, and the obtained plants are mostly chimeras with different ploidy, so the period is longer, the workload is large, and the cost is higher.

In addition, Huang Guo Zheng et al (new species of lotus, China flower horticulture, 18: 46-49, 2007) obtain a part of triploid plant by inducing the lotus seed to double, but do not disclose a specific processing flow.

Disclosure of Invention

The invention aims to provide a method for breeding lotus triploid by 2n pollen hybridization, which can efficiently generate 2n pollen by inducing lotus stamen, and then carry out sexual hybridization with diploid pollen to obtain offspring, wherein the obtained triploid plant has large shape, improved flower quantity, increased flower height, bright flower color, simple and convenient breeding process operation and short breeding time, can simultaneously treat flower buds on a large scale to efficiently induce large batch of 2n pollen, has extremely low influence on self pistil group, and can develop self-pollination after blooming to carry out triploid inbred line breeding with specific requirements.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for breeding lotus triploid by 2n pollen hybridization comprises the following steps:

1) induction of 2n pollen

Selecting a lotus flower bud which is robust in growth, 3-3.5 cm in length and milky-white in stamen group, poking outer petals of the flower bud in the morning, only reserving 4-6 petals of the innermost layer to wrap the stamen group, filling cotton matter between the poked outer petals and the reserved inner petals, wherein the filling thickness is 3-4 mm, dropwise adding 2-3 ml of colchicine solution with the concentration of 0.02-0.03 wt.% into the cotton matter to enable the cotton matter to be saturated with water, resetting the poked outer petals and inducing;

after inducing for 20-30 hours, dripping colchicine solution with the concentration of 0.02-0.03 wt.% again until the cotton is saturated with water, inducing for 20-30 hours, removing the cotton, and taking pollen after the pollen of the flowers is loosened;

2) artificial pollination for hybridization

Collecting the stamen group of loose powder in the step 1) as a male parent, selecting a lotus flower on the day 2 of blooming as a female parent, sleeving a tuck net on the female parent before blooming, removing the male parent, clamping the loose powder stamens, smearing the pistil stigma of the female parent before 10 am, and sleeving the tuck net back after finishing;

and (5) after pollination for 25-30 days, the lotus seedpod grows mature, the lotus seeds are brown or black brown, and the lotus seeds are peeled off.

Further, in the step 1), 8-10 days after cotton substances are removed, the treated buds grow mature and are about to bloom, the buds are sleeved with a string bag, 2-3 days later, petals of the mature buds are gradually opened, 4-7 points in the morning of the day 2 of blooming, anthers of pistil groups are cracked, and a large amount of pollen is scattered.

Preferably, the cotton substance is cotton wool, cotton or cotton yarn.

In the step 1), the induction time is 20-25 hours each time.

Further, in the step 1), after the opened outer petals are reset, a paper bag is sleeved on the flower buds.

And in the step 2), collecting loose stamens and putting parchment paper in the pollen at the 2 nd day of blossom of the treated buds and between 7 and 8 o' clock in the morning when the male parents are collected.

Preferably, the flower bud is an american lotus flower bud.

In the invention, the lotus stamen is induced to efficiently generate 2n pollen, and sexual hybridization is carried out on the pollen and diploid pollen to obtain the triploid lotus seed. When an explant is selected, a flower bud with the length of 3-3.5 cm is selected, the flower bud is in a period when anthers are white mainly and slightly yellowish, the period is approximately a primary spermatocyte period, spermatogonium is subjected to first meiosis, cotton substances are wrapped outside innermost petals and then are soaked in colchicine for treatment, liquid permeates through the epidermis and the endothelium of the innermost petals and the anthers after 10-15 hours and finally reaches a pollen sac, the pollen sac develops to a secondary spermatocyte period at the time and is subjected to second meiosis, the colchicine has the effect of inhibiting or damaging the formation of spindle microtubules, secondary spermatocytes containing two staining monomers cannot normally complete second meiosis, and the formation of 2n pollen is induced. The method selects the lotus buds in the optimal period for inducing and generating 2n pollen for induction treatment by the combination judgment of the length of the lotus buds and the color of anther so as to improve the induction success rate of the 2n pollen.

Before inducing 2n pollen, the cotton substance is wrapped and clamped by a layer of petals at the innermost side, a layer of petals is similarly added, the cotton substance is not directly contacted with a pistil group and is not directly exposed outside a flower bud, so that colchicine immersed in the cotton substance can slowly permeate into the pistil group by retaining the petals at the inner layer, the development of the pistil group is not greatly influenced, the good development of the pollen is ensured, and the induction rate of the 2n pollen is improved; the outer layer is also protected by petals, and the cotton substance absorbs the colchicine liquid with small water evaporation amount, so that the concentration of the colchicine liquid is not gradually increased due to evaporation, or the total amount of the liquid is reduced due to evaporation, and the permeation effect to the pistil group in the inner layer is not influenced.

Plant 2n pollen doubling can only be spread out during the secondary spermatocyte stage, this window being only about 2 days for the whole androecium of the lotus, and it is therefore necessary to screen for suitable colchicine concentrations to achieve the best 2n pollen induction during this stage. The method combines the filling mode of the cotton substance, 0.02-0.03 wt.% of colchicine solution is dripped on the absorbent cotton to the lotus buds which are 3-3.5 cm in length and are in the secondary spermatocyte period, the colchicine liquid soaked in the cotton substance slowly permeates into gynoecium through the reserved petals of the inner layer, the concentration is proper and stable, the development of the cotton substance cannot be greatly influenced, the good development of the pollen is ensured, and the induction rate of 2n pollen is improved.

Furthermore, colchicine treatment on the lotus requires proper treatment time combined with concentration control, gynoecium development is caused if the treatment time is too long, and most gynoecium is rotten and dead if the treatment time exceeds 3 days under the treatment concentration of the invention through tests.

When the flower buds are mature but not opened, the bee cap is sleeved with the nylon string bag to prevent the bee from visiting the flower and generating pollen pollution of other flowers when the flower blooms, and the bee cap is removed 2 days after pollination, namely 3 days after the flower blooms, at the moment, pollen enters ovules and the pollination ability of the stigma is extremely low.

The cotton substance is preferably absorbent cotton, the absorbent cotton is easy to absorb water, can keep a certain shape after absorbing the water, has better water retention property, and is easy to obtain as an industrial product.

Compared with the prior art, the invention has at least the following beneficial effects:

by utilizing the method, the source of the pollen of the male parent is wide, the pollen can be any target lotus variety, whether the variety has a plurality of petals or the variety has thick petals, the variety is easily treated and induced by the method, the doubled 2n pollen is obtained, and then the pollen is sexually crossed with the diploid plant; the 2n pollen has an induction rate of 30% or more when treated with 0.02 wt.% colchicine solution, and the 2n pollen has an induction rate of 25% or more when treated with 0.03 wt.% colchicine solution; the method has the advantages of good repeatability and high stability, and compared with the female parent, the obtained triploid plant has large plant type, obviously increased flower quantity, increased flower height and bright flower color.

The method can process the flower buds in a large scale in the flower season to obtain a large amount of 2n pollen, the pollen is taken and pollinated for hybridization or selfing after the flower blooms, the seeds collected in autumn of the year contain triploid seeds, and the breeding time in the prior art is greatly shortened. As for the lotus, the process of treating buds by colchicine, blooming, pollinating and harvesting is only 35 days at the fastest speed, the breeding period is greatly shortened, the diversity of triploid germplasm resources of the lotus is increased, and good germplasm resources are reserved for breeding new and excellent lotus varieties.

Drawings

FIG. 1 is a graph of the American lotus bud length comparison of the inventive and comparative examples.

Fig. 2 is a diagram of the bud treatment process by colchicine solution in the present invention, wherein A, B, C, D represents different treatment steps.

FIG. 3 is a diagram of the procedure for the treatment of flower buds with a colchicine solution in a comparative example, wherein A, B, C, D represents the different treatment steps, respectively.

FIG. 4 is a pollen scattering diagram of pollen according to an embodiment of the present invention.

FIG. 5 is a diagram of artificial pollination in an example of the invention.

FIG. 6 is a diagram of the flow cytometry detection result of the young leaves of the survived plants in the example of the present invention, wherein A is a diploid of the 'Weishan red' lotus, B is a triploid of the filial generation of the example, and C is a natural triploid.

FIG. 7 is a diagram showing the chromosome counting observation result of the root tip of the double plant in the embodiment of the present invention, wherein a is a diploid of the 'Weishan red' lotus, b is a triploid of the filial generation of the embodiment, and c is a natural triploid.

Detailed Description

The present invention is further illustrated by the following specific examples.

EXAMPLES AND COMPARATIVE EXAMPLES selection of Lotus triploid Using 2n pollen hybridization

1. Induction of 2n pollen

American lotus buds with the length of 3-3.5 cm are respectively taken as treatment objects of the embodiment group of the invention, American lotus buds with the length of 2.0cm are taken as treatment objects of the comparative example group, and the treatment objects are shown in figure 1.

Except that the mode of wrapping the absorbent cotton and the dropping liquid amount are different, if no special description is provided, other operations are consistent:

selecting a bud with the length of 3-3.5 cm, wherein anthers are milky white and slightly yellowish, the bud is approximately in the primary spermatocyte period, slightly poking outer petals of the bud (figure 2A) to enable only 1 layer of 4-6 petals on the inner side to wrap a stamen group (figure 2B), inserting 1 layer of absorbent cotton with the thickness of 3-4 mm between the bud and the stamen group (figure 2C) by using blunt-ended tweezers, dropwise adding 2ml of colchicine liquid with corresponding concentration (table 1) by using a liquid transfer gun, resetting the poked outer petals to an original state (figure 2D), and finally sleeving a paper bag to prevent the colchicine liquid from evaporating too fast; and (3) dropwise adding 2ml of colchicine liquid at 7-10 points in the next morning, removing paper bags and absorbent cotton at 7-10 points in the 3 rd morning, and repeatedly treating 3 buds at each concentration.

Selecting a flower bud with the length of 2.0cm and strong growth, wrapping 3 layers of the flower bud by using absorbent cotton at 7-10 am, externally winding a cotton thread for fixation (figure 3A), then dropwise adding 1ml of colchicine liquid with corresponding concentration (figure 3B) (table 1) by using a liquid transfer gun, and finally sequentially sleeving a cut plastic bag and a paper bag (figures 3C and 3D), wherein the flower bud is easily heated by only sleeving one layer of the plastic bag, and only the paper bag directly contacts the absorbent cotton to absorb water; dripping 1ml colchicine liquid at 7-10 point in the next morning, removing the cover bag and absorbent cotton at 7-10 point in the 3 rd morning, and treating 3 flower buds at each concentration.

8-10 days after the absorbent cotton is removed, namely when the flower buds are mature and not opened, the nylon net bag is sleeved to prevent the bees from visiting the flowers when the flowers are opened and pollen pollution of other flowers is caused. After 2-3 days, the petals of the mature flower buds are gradually opened, a large amount of pollen is scattered, referring to fig. 4, on the day 2 of blooming, a large amount of pollen is scattered by anthers of stamen groups, and each anther can be scattered into thousand pollen grains.

TABLE 1 colchicine gradient and microscopic examination results for inducing 2n pollen of American lotus

2. Artificial pollination for hybridization

Since most stamens are deformed after the 2 cm-long buds are treated by colchicine, part of the flowers even die directly without expanding, and sufficient pollen cannot be obtained in the later period, the flowers formed by the developed buds of 3-3.5 cm are only selected in the subsequent experiments to collect pollen for artificial pollination.

Collecting the loose powder stamens between 7-8 points in the morning on day 2 of blooming, selecting female parents on day 2 of blooming, sleeving nylon net bags on the female parents 1-2 days before blooming, castrating, picking up the net bags, clamping 10-15 American lotus loose powder stamens by using tweezers, smearing female parent receptacle stigma for 5-6 times (figure 5), and finally sleeving back the net bags.

Removing the net bag 2 days after pollination, growing the receptacle (lotus seedpod) to be mature 25-30 days after pollination, peeling off the lotus seed, and storing at 4 degrees.

3. Accelerating germination and cultivating

And carrying out conventional pregermination and cultivation management on the harvested lotus seeds.

As a result, it was found that: triploid seedlings could not be detected in the hybrid lotus seeds of pollen obtained by 0.01 wt.% concentration treatment, whereas stamens of treated buds at 0.05 wt.% concentration are often black dead and abortive and pollination fructification rate is low.

Pollen obtained from flower buds treated with 0.02 wt.% concentration is subjected to artificial pollination for 6 hybrid combinations to obtain 50 flowers, and 108 mature seeds are collected. In the next year, 50 combinations are randomly selected for accelerating germination and cultivation, and conventional maintenance management is carried out to obtain 28 plants (table 2).

TABLE 2 colchicine 0.02 wt.% concentration induces the crossing effect of 2n pollen

3. Ploidy identification

Young leaves of 28 surviving plants were picked and subjected to flow cytometry (see results in fig. 6A, 6B, 6C) to obtain 3 triploid plants (table 2) accounting for 11%, having material numbers of G178, B3 and B4, respectively, and belonging to combinations of M360 × M516(G178), M305 × M423(B3 and B4), respectively. Chromosome counting observation was performed on the root tips of the doubled plants (fig. 7a, 7b, 7c), and it was confirmed that all three were triploid plants. Due to random pregermination, the number of sowed seeds is small, and the remaining 4 combinations do not obtain triploid plants in the pregermination, but the possibility that the remaining 58 hybrid lotus seeds contain triploid lotus seeds is not excluded.

The obtained 3 triploid plants have the same chromosome number as natural triploid lotus plants in the day of sunny day, have no big difference in appearance, and have 3 longer chromosomes, and the diploid has 2 longest chromosomes.