A method for the hybridization breeding of diploid cultivars of musa acuminata

Abstract

The application relates to a method for diploid cultivated banana hybridization breeding, and belongs to the technical field of hybridization breeding. The application improves the seed setting rate of hybridization through parent selection and a proper pollination method, and improves the hybrid embryo plantlet rate through embryo in-vitro culture, and further improves the survival rate of hybridization. The method can improve the seed number of a single fruit from almost zero to 0.72, has the characteristics of simple operation, high hybridization efficiency, high seed setting rate and high seed plantlet rate, overcomes the problem of high sterility of diploid cultivated banana, solves the problems of poor hybridization compatibility, difficult seed setting and low seed germination rate of diploid cultivated banana, and has important significance for banana hybridization breeding technology. The application solves the problem of hybridization of diploid cultivated banana with genotype AA, and opens up a new method, way and technical support for new variety cultivation of Gongjiao (AA), Xiangyajiao (AAA) and Longyajiao (AAB).

Description

Technical Field

[0001] This invention relates to the field of hybridization breeding technology, and in particular to a method for hybridization breeding of diploid cultivated bananas. Background Technology

[0002] Bananas (Musa spp.) are the world's largest importer and exporter of fresh fruit, and also an important food crop in tropical and subtropical developing countries. China is one of the oldest countries in the world to cultivate bananas, and is the world's second largest producer and consumer of bananas after India. It is reported that my country has abundant banana germplasm resources and a wide variety of species. Cultivated bananas mainly include the *Musa acuminata*, *Musa balbisiana*, *Musa itinerans*, *Musa basjoo*, *Musa nagensium*, and *Musa coccinea*. Cultivated banana species mainly evolved from hybrids within or between the two primitive wild banana species, *Musa acuminata* (genus A) and *Musa balbisiana* (genus B). Most cultivated bananas are triploid and highly sterile, while a few are seedless diploids or tetraploids. Diploid cultivated bananas are intersubspecies hybrids with characteristics such as high sterility, parthenocarpy, and asexual reproduction, making banana hybridization breeding work very difficult. Studies have found that a small number of diploid cultivated varieties have weak fertility and can produce a small number of seeds when hybridized with wild bananas.

[0003] In banana wide hybridization, different banana hybrid combinations show significant differences. Plantains (ABB) and bananas produce a large number of seeds, and a small number of these seeds germinate and grow normally. However, bananas of the AAA, AAB, and AA types almost never produce seeds. Among these, the diploid cultivated banana 'Rose Banana' of genotype AA only yields a few seeds from a few plants, but none germinate successfully (Li Weiming et al., 2021). This demonstrates that diploid cultivated banana hybridization suffers from extremely low seed production, abnormal seed development, and extremely low germination rates. Regarding the extremely difficult problem of hybridization breeding of diploid cultivated bananas of genotype AA, there are currently no successful reports in China.

[0004] The seed setting rate of bananas can be increased by selecting appropriate hybridization combinations and improving hybridization pollination methods (Zhao Ming et al., 2019). For example, patent CN109924123A, "A Hybridization Pollination Method to Improve the Seed Setting Rate of Bananas", uses triploid banana (ABB) as the female parent and wild banana as the male parent. Among them, the banana 'Jinfen No. 1' is used as the female parent material and the wild banana 'LW' is selected as the male parent material. Through repeated pollination, the fertilization probability is increased. Pollination is carried out at the middle comb position of the female flower of the female parent. Pollination is carried out at the stage when the female flower bracts are obviously open, the opening angle between the bracts and the rachis is 10° to 80°, and the stigma is exposed. The average number of seeds per fruit can reach 1.98. This provides technical support for the hybridization breeding of bananas and the cultivation of new varieties such as bananas. However, it is not applicable to the hybridization breeding of diploid cultivated bananas.

[0005] Therefore, it is essential to develop a method to improve the seed setting rate of diploid banana hybrids, as well as the seedling and mature plant rates of offspring, thereby solving the problems of difficult seed setting and low seed germination rate in diploid banana hybrids. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for hybrid breeding of diploid bananas with high hybridization efficiency, high seed setting rate, and high embryo-to-plant line rate.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for hybrid breeding of diploid cultivated bananas, comprising the following steps:

[0009] S1. Select diploid cultivated bananas with fertile pollen as the female parent, and select wild bananas or diploid cultivated bananas with fertile pollen as the male parent.

[0010] S2. Artificially hybridize the selected female and male parents, spray pollen germination medium after artificial pollination, and harvest hybrid seeds when the fruit matures.

[0011] S3. Take the embryos of the hybrid seeds for in vitro culture. The rooted seedlings obtained from the in vitro culture are transplanted and hardened off to obtain hybrid plants.

[0012] The pollen germination medium consists of an aqueous solution containing 0.01 g / L H3BO3, 0.25 g / L MgSO4·7H2O, 0.25 g / L KNO3, 0.4 g / L Ca(NO3)2, and 12%–18% glucose by mass, with a pH of 5.8.

[0013] This invention improves the seed setting rate of hybrids by selecting specific parents and combining pollination with spraying pollen germination medium. At the same time, it improves the hybrid embryo line formation rate by combining in vitro embryo culture, thereby improving the survival rate of hybrids.

[0014] In a preferred embodiment of the method described in this invention, the fertile pollen is mature, viable, and capable of germination produced by male flowers. Pollen viability can be detected using conventional methods, such as MTT staining or TTC staining. Pollen that stains darkly with MTT (2,5-diphenyltetrazolium bromide, thiazolyl blue) or TTC (2,3,5-triphenyltetrazolium chloride) is considered viable.

[0015] As a preferred embodiment of the method described in this invention, the diploid cultivated banana is a cultivated banana with a chromosome ploidy of diploidity and a genotype of AA.

[0016] In a preferred embodiment of the method described in this invention, the diploid cultivated banana with fertile pollen is a rose banana.

[0017] In a preferred embodiment of the method described in this invention, the wild banana is a pointed-leaf banana; preferably, the pointed-leaf banana includes Calcutta 4 (M. acuminata ssp. burmannicoides) or Tropical Banana simonii. The Tropical Banana simonii is a small-fruited banana introduced from the Chinese Academy of Tropical Agricultural Sciences.

[0018] In a preferred embodiment of the method described in this invention, the banana plant is a tropical banana (Musa acuminata).

[0019] In a preferred embodiment of the method described in this invention, in step S1, wild bananas are selected as the parent plant.

[0020] In a preferred embodiment of the method described in this invention, in step S1, the pointed-leaf banana is selected as the male parent. Preferably, the tropical small-fruited banana is selected as the male parent. The tropical small-fruited banana is a small-fruited banana introduced from the Chinese Academy of Tropical Agricultural Sciences.

[0021] As a preferred embodiment of the method described in this invention, the artificial pollination is carried out during the warm and humid season from March to June or from mid-August to October.

[0022] In a preferred embodiment of the method described in this invention, the artificial pollination is carried out at night or in the morning when the male flowers open, the anthers mature, split open, and the pollen is exposed, and the pollen is collected; at night or in the morning when the female flower bracts open and the stigma of the female flower is exposed, the pollen is applied to the stigma to complete the artificial pollination.

[0023] In a preferred embodiment of the method described in this invention, the artificial pollination is carried out at night when the female flower bracts open and the stigma of the female flower is exposed, by applying pollen to the stigma to complete the artificial pollination.

[0024] In a preferred embodiment of the method described in this invention, the pollinating combs used for artificial pollination are the 1st to 3rd combs.

[0025] In a preferred embodiment of the method described in this invention, the pollinating comb used for artificial pollination is the first comb.

[0026] In a preferred embodiment of the method described in this invention, the fruit is matured to the point where it has more than 50% flesh and most of the seeds have blackened seed coats.

[0027] As a preferred embodiment of the method described in this invention, the method for harvesting the hybrid seeds is as follows: after the fruit matures and softens, the pulp is removed, and then the seeds are placed in a sealed space for fermentation. After the pulp on the seed coat is fully removed, empty and shriveled seeds are removed and then the seeds are dried.

[0028] As a preferred embodiment of the method described in this invention, the method for obtaining the embryo of the hybrid seed is as follows: after the seed is disinfected, the seed coat is broken by squeezing and the embryo is peeled off.

[0029] In a preferred embodiment of the method described in this invention, the seed disinfection is performed by sterilizing the seed surface using alcohol and sodium hypochlorite.

[0030] As a preferred embodiment of the method described in this invention, the specific method for seed disinfection is as follows: In a clean bench, the seeds are soaked in 70-75% alcohol for 30-60 seconds, then transferred to 2-3% sodium hypochlorite for disinfection for 15-20 minutes, then rinsed with sterile water 3-5 times, and finally dried with sterile filter paper.

[0031] In a preferred embodiment of the method described in this invention, the embryo removal is performed in a clean bench by using sterile tweezers to hold the seed in the gap of sterile nail clippers, pressing the end of the clippers slightly to squeeze the seed and break the seed coat, thus splitting the seed in two and removing the embryo.

[0032] As a preferred embodiment of the method described in this invention, the in vitro culture specifically involves: inoculating the embryo of a hybrid seed into an induction medium for dark culture to obtain a bud, then inoculating the bud into a proliferation medium to propagate it into a cluster of buds, and then inoculating the cluster of buds into a rooting medium to root them, thereby obtaining rooted seedlings.

[0033] In a preferred embodiment of the method described in this invention, the dark culture time is at least one week.

[0034] As a preferred embodiment of the method described in this invention, the induction culture medium consists of: MS medium, 6-benzylaminopurine (6-BA) 2-3 mg / L, KT kinetin (6-furfurylaminopurine) 1-2 mg / L, adenine 2.5-5 mg / L, agar 4 g / L, sucrose 30 g / L, and pH 5.8-6.

[0035] The proliferation medium consisted of: MS medium, 6-benzylaminopurine (6-BA) 2.5-5 mg / L, naphthaleneacetic acid (NAA) 0.1-0.2 mg / L, agar 4 g / L, sucrose 30 g / L, and pH 5.8-6.

[0036] The rooting medium consists of: 1 / 2 MS medium, indolebutyric acid (IBA) 0.5-2 mg / L, naphthaleneacetic acid (NAA) 0.1-0.2 mg / L, 0.5% activated carbon, agar 4 g / L, and pH 5.8-6.

[0037] As a preferred embodiment of the method described in this invention, the induction culture medium consists of: MS medium, 6-benzylaminopurine (6-BA) 3 mg / L, KT kinetin (6-furfurylaminopurine) 2 mg / L, adenine 2.5 mg / L, agar 4 g / L, sucrose 30 g / L, and pH 6.0.

[0038] The proliferation medium consisted of: MS medium, 6-benzylaminopurine (6-BA) 4 mg / L, naphthaleneacetic acid (NAA) 0.1 mg / L, agar 4 g / L, sucrose 30 g / L, and pH 6.0.

[0039] The rooting medium consisted of: 1 / 2 MS medium, indolebutyric acid (IBA) 1 mg / L, naphthaleneacetic acid (NAA) 0.2 mg / L, 0.5% activated carbon, agar 4 g / L, and pH 6.0.

[0040] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0041] This invention provides a method for hybrid breeding of diploid cultivated bananas. By selecting parents and using appropriate pollination methods, the seed-setting rate of hybrids is improved. Simultaneously, by combining in vitro embryo culture, the rate of hybrid embryos forming new plants is increased, thereby improving the survival rate of hybrids. The method provided by this invention can increase the seed yield of diploid cultivated bananas from almost zero to an average of 0.72 seeds per fruit, with a seedling survival rate of 2%. It features simple operation, high hybridization efficiency, high seed-setting rate, and high seed-to-plant ratio, overcoming the problem of high sterility in diploid cultivated bananas and solving problems such as poor hybridization compatibility, difficulty in seed setting, and low seed germination rate. This invention is of great significance to banana hybrid breeding technology. By solving the hybridization problem of diploid cultivated bananas with genotype AA, this invention opens up new methods, pathways, and technical support for the breeding of new varieties such as Gongjiao (AA), Xiangya (AAA), and Longya (AAB). Attached Figure Description

[0042] Figure 1 The images show the male flowers of the rose banana plant in Example 7 of this invention (A), the MTT staining of its pollen (B), and the germination of its pollen in PGM liquid medium containing 12% glucose (C).

[0043] Figure 2 The seeds are obtained from the hybrid combination of 'Rose Banana' and 'Tropical Small-fruited Wild Banana' in Example 7 of this invention;

[0044] Figure 3 Examples of the present invention 7 show the following: the embryos of the seeds obtained from the hybrid combination of 'Rose Banana' and 'Tropical Small Fruit Banana' differentiate into buds on the induction medium (A); the buds are propagated into cluster buds on the proliferation medium (B); and the cluster buds are rooted on the rooting medium to form rooted seedlings (C).

[0045] Figure 4 This describes the secondary seedlings obtained after transplanting seedlings from the hybrid combination of 'Rose Banana' and 'Tropical Small Fruit Banana' in Example 7 of this invention. Detailed Implementation

[0046] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0047] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0048] During the flowering period of male flowers, observe whether the anthers of diploid cultivated banana male flowers can naturally dehisce to produce mature pollen. Then, use the MTT or TTC staining method to determine pollen viability and use the in vitro germination method to determine pollen germination. Pollen that is viable and can germinate is fertile pollen.

[0049] When the fruit is ripe, it has more than 50% flesh and most of the seeds have blackened seed coats.

[0050] Tropical wild banana: a wild banana variety introduced from the Chinese Academy of Tropical Agricultural Sciences; Yunnan wild banana: a wild banana variety originating from Xishuangbanna, Yunnan.

[0051] Example 1: Screening of pollen germination culture medium

[0052] Pollen germination in vitro:

[0053] Pollen from 'Tropical Banana davidii' (pointed-leaf banana) and 'Napo Banana davidii' (long-stemmed banana) was cultured in vitro using pollen germination medium (PGM liquid medium) containing different concentrations of glucose.

[0054] Pollen germination medium 1 (PGM liquid medium containing 6% glucose): 0.01g H3BO3, 0.25g MgSO4·7H2O, 0.25g KNO3, 0.4g Ca(NO3)2, 6% glucose, 1L water, pH 5.8.

[0055] Pollen germination medium 2 (PGM liquid medium containing 12% glucose): 0.01g H3BO3, 0.25g MgSO4·7H2O, 0.25g KNO3, 0.4g Ca(NO3)2, 12% glucose, 1L water, pH 5.8.

[0056] Pollen germination medium 3 (PGM liquid medium containing 18% glucose): 0.01g H3BO3, 0.25g MgSO4·7H2O, 0.25g KNO3, 0.4g Ca(NO3)2, 18% glucose, 1L water, pH 5.8.

[0057] Pollen germination medium 4 (PGM liquid medium containing 24% glucose): 0.01g H3BO3, 0.25g MgSO4·7H2O, 0.25g KNO3, 0.4g Ca(NO3)2, 24% glucose, 1L water, pH 5.8.

[0058] Four different pollen germination media were sprayed onto the pollen of 'Tropical Small-fruited Banana' (pointed-leaf banana) and 'Napo Wild Banana' (long-stemmed banana), respectively, and incubated at 30℃ for 12 hours. The germination rate of the pollen was observed under a 40×10x optical microscope. The germination standard was defined as the pollen tube length exceeding twice the pollen grain diameter. The number of germinated pollen was recorded, and the pollen germination rate was calculated according to the following formula:

[0059] Pollen germination rate (%) = Number of germinated pollen in the field of view / Total number of pollen in the field of view * 100%.

[0060] The effects of pollen germination medium containing different concentrations of glucose on pollen germination are shown in Table 1 below.

[0061] Table 1

[0062]

[0063] Table 1 shows that the pollen of 'Radermachera repens' and 'Napo wild banana' was germinated most readily on pollen germination media containing 12% and 18% glucose concentrations, which is more conducive to pollen germination.

[0064] Example 2

[0065] Using 'Musa acuminata' (AA) banana as the female parent and 'Calcutta 4' (M. acuminatassp. burmannicoides, AA), 'Treco dauber basilica' (pointed-leaf banana), and 'Napo basilica' (long-stemmed banana) as the male parents, artificial cross-pollination was performed on the entire bunch of bananas in the evening when the bracts opened from August to October. Immediately after pollination, the entire bunch was sprayed with PGM liquid medium containing 12% glucose as described in Example 1, and allowed to ripen. After the fruit ripened and the pulp softened, the banana peel was removed, and the pulp was cut open or peeled apart by hand to obtain the seeds containing pulp. These seeds were placed in a sealed plastic bag for natural fermentation for 2-3 days, then placed in a mesh bag and rubbed to completely separate the seeds from the pulp. Empty and shriveled seeds were removed by water flotation, and the seeds were placed in a ventilated place to dry the surface moisture and counted.

[0066] Observations revealed that the 'Jiali' banana lacked mature, fertile pollen, and no seeds were harvested when the fruit matured.

[0067] Example 3

[0068] Using 'Haigong' (Haigong banana) as the female parent and 'Calcutta 4' (M. acuminatassp. burmannicoides, AA), 'Treco small-fruited wild banana' (pointed-leaf banana), and 'Napo wild banana' (long-stemmed banana) as the male parents, artificial cross-pollination was carried out on the entire bunch of bananas in the evening when the bracts opened from August to October. Immediately after pollination, the entire bunch was sprayed with PGM liquid medium containing 12% glucose as described in Example 1, and the fruit was allowed to mature. The method for obtaining the seeds was the same as in Example 2.

[0069] Observations revealed that the 'Haigong' banana variety lacked mature, fertile pollen, and no seeds were harvested when the fruit matured.

[0070] Example 4

[0071] Using 'Bubang Canna' (canna lily) as the female parent, and 'Calcutta 4' (M. acuminatas sp. burmannicoides, AA), 'Treco dauber basilica' (pointed-leaf banana), and 'Napo basilica' (long-stemmed banana) as the male parents, artificial cross-pollination was performed on the entire bunch of bananas in the evening when the bracts opened in August and September. Immediately after pollination, the entire bunch was sprayed with PGM liquid medium containing 12% glucose as described in Example 1, and the fruit was allowed to mature. The method for obtaining the seeds was the same as in Example 2.

[0072] Observations revealed that the Bubang canna lily had no mature, fertile pollen, and no seeds were harvested when the fruit matured.

[0073] Example 5

[0074] Using 'Menglun Beauty Canna' (canna lily) as the female parent and 'Calcutta 4' (M. acuminatas sp. burmannicoides, AA), 'Treco Small-fruited Wild Banana' (pointed-leaf banana), and 'Napo Wild Banana' (long-stemmed banana) as the male parents, artificial cross-pollination was performed on the entire bunch of bananas in April and from August to October when the bracts opened at night. Immediately after pollination, the entire bunch was sprayed with PGM liquid medium containing 12% glucose as described in Example 1, and the fruit was allowed to mature. The method for obtaining the seeds was the same as in Example 2.

[0075] Observations revealed that 'Menglun Canna' lacked mature, fertile pollen, and no seeds were harvested when the fruit matured.

[0076] Example 6

[0077] Using 'Buddha's Hand Banana' (Calamus nigra) as the female parent and 'Tropical Small-fruited Wild Banana' (Calamus acuminate) as the male parent, artificial cross-pollination was performed on the entire bunch of bananas in the morning of October. Immediately after pollination, the entire bunch was sprayed with PGM liquid culture medium containing 12% glucose as described in Example 1, and the fruit was allowed to mature. The method for obtaining the seeds was the same as in Example 2.

[0078] Observations have revealed that 'Buddha's Hand Banana' can produce mature pollen, and when the fruit matures, the seeds can be harvested.

[0079] Example 7

[0080] Using 'Rose Banana' as the female parent and 'Calcutta 4' (M. acuminata ssp. burmannicoides, AA, pointed-leaf banana), 'Tropical Small-fruited Wild Banana' (pointed-leaf banana), 'Yunnan Small-fruited Wild Banana' (pointed-leaf banana), 'Rose Banana,' 'Napo Wild Banana' (long-stemmed banana), and 'Pisang Klutuk Wulung Wild Banana' (abbreviated as 'PKW', BB) as the male parent, artificial cross-pollination was performed on the entire bunch of bananas in the evening or morning of April and September-October. Immediately after pollination, the entire bunch was sprayed with PGM liquid medium containing 12% glucose as described in Example 1, serving as the experimental group. The crosses between 'Rose Banana' and 'Tropical Small-fruited Wild Banana', and between 'Rose Banana' and 'Rose Banana', served as control groups without spraying the PGM liquid medium containing 12% glucose as described in Example 1, until the fruit matured. The method for obtaining seeds was the same as in Example 2. The average number of seeds per fruit was calculated based on the number of seeds obtained using the following formula:

[0081] Average number of seeds per fruit = number of seeds / pollination fruit index * 100.

[0082] Observations revealed that 'Rose Canna' produces mature, fertile pollen. The male flowers of Rose Canna (A), their pollen MTT staining (B), and the germination of their pollen in PGM liquid medium containing 12% glucose (C) are shown below. Figure 1 As shown.

[0083] When the fruit matured, seeds were obtained in all experimental groups. In the control group, the hybrid combination of 'Rose Banana' and 'Tropical Small Fruit Banana' obtained seeds, while the hybrid combination of 'Rose Banana' and 'Rose Banana' did not obtain seeds.

[0084] Seeds obtained from the hybridization of 'Rose Banana' and 'Tropical Small-fruited Wild Banana' are as follows Figure 2 As shown.

[0085] Example of embryo culture

[0086] Seeds harvested in Examples 6 and 7 were used to separate embryos for in vitro embryo culture.

[0087] (1) Embryo removal

[0088] Take the seeds that have been dried to remove surface moisture and set them aside. If they cannot be processed immediately, they need to be dried for 3-5 days. After drying, store the seeds in a cool, dry environment.

[0089] In a clean bench, soak the seeds in 70-75% alcohol for 30-60 seconds, then transfer them to 2-3% sodium hypochlorite for 15-20 minutes for sterilization. Rinse them 3-5 times with sterile water, and finally blot dry with sterile filter paper. Then, use sterile tweezers to hold the seed in the gap of sterile nail clippers, and gently press the end of the clippers to break the seed coat, separating the seed in two and removing the embryo. Calculate the embryo yield using the formula: Embryo recovery rate (%) = (Number of embryos / Number of seeds) * 100.

[0090] (2) Embryo culture

[0091] Induction medium: MS medium, 6-benzylaminopurine (6-BA) 3 mg / L, KT kinetin (6-furfurylaminopurine) 2 mg / L, adenine 2.5 mg / L, agar 4 g / L, sucrose 30 g / L, pH 6.0, sterilized at 121℃ for 20-30 minutes.

[0092] Proliferation medium: MS medium, 6-benzylaminopurine (6-BA) 4 mg / L, naphthaleneacetic acid (NAA) 0.1 mg / L, agar 4 g / L, sucrose 30 g / L, pH 6.0, sterilized at 121℃ for 20-30 minutes.

[0093] Rooting medium: 1 / 2 MS medium, indolebutyric acid (IBA) 1.0 mg / L, naphthaleneacetic acid (NAA) 0.2 (0.1-0.2) mg / L, 0.5% activated carbon, agar 4 g / L, pH 6.0, sterilized at 121℃ for 20-30 minutes.

[0094] Embryos were inoculated into an induction medium and cultured in the dark for 15-20 days. The embryos differentiated into buds, which were then inoculated into a proliferation medium to propagate bud clusters. These clusters were then inoculated into a rooting medium to root, resulting in rooted seedlings (hybrid plantlets). The following diagram illustrates the differentiation of embryos from seeds obtained from a hybrid of 'Rose Banana' and 'Tropical Small-fruited Wild Banana' on an induction medium (A), the propagation of buds into bud clusters on a proliferation medium (B), and the rooting of these bud clusters into rooted seedlings on a rooting medium (C). Figure 3 As shown.

[0095] After a week of hardening-off in a greenhouse, the rooted seedlings are transplanted into nutrient cups and grown for about two months to obtain secondary seedlings with 7-8 leaves. These can then be transplanted to the field or used for experiments such as disease resistance identification. The secondary seedlings obtained after transplanting seedlings from a hybrid of 'Rose Banana' and 'Tropical Small-fruited Wild Banana' are shown below. Figure 4 As shown.

[0096] Embryo survival rate (%) = (Number of surviving embryos / Number of embryos) * 100;

[0097] Embryo success rate (%) = Plant coefficient / Number of surviving embryos * 100;

[0098] Number of viable embryos: The number of viable embryos that can swell, become villous, sprout, grow roots, and develop callus tissue on the induction medium; Strain number: All the secondary seedlings obtained from one embryo that can produce a secondary seedling are called a strain.

[0099] Table 2 below shows the average number of seeds per fruit, embryo yield, embryo survival rate, and embryo-to-plant line rate for different hybridization combinations in Examples 6 and 7.

[0100] Table 2

[0101]

[0102]

[0103] Note: All results were obtained by artificial cross-pollination of whole bunches of bananas, and the statistical results include those of whole bunches of bananas.

[0104] Table 2 shows that the average number of seeds per fruit in the hybrid combination of 'Buddha's Hand Banana' and 'Tropical Small Fruit Banana' was 0.14, but the seed embryo yield was 0, and no plants were obtained.

[0105] The hybrids using 'Rose Banana' as the female parent and 'Tropical Small-fruited Wild Banana', 'Yunnan Small-fruited Wild Banana', 'Rose Banana', and 'Napo Wild Banana' as the male parents produced a high number of seeds. Embryos were obtained from hybrid seeds using 'Calcutta 4', 'Tropical Small-fruited Wild Banana', and 'Rose Banana' as the male parents. The hybrid of 'Rose Banana' and 'Tropical Small-fruited Wild Banana' had the highest embryo yield, producing hybrid plants with an embryo survival rate of 5.56% and an embryo-to-plant line rate of 100%. The calculated seedling rate of the hybrid seeds was 2.27%. Compared to not spraying with PGM liquid medium containing 12% glucose, spraying immediately after pollination was more beneficial in increasing the number of seeds and the embryo yield.

[0106] 3. The distribution of seeds from different comb numbers of the hybrid combinations of 'Rose Banana' and 'Tropical Small-fruited Wild Banana' was statistically analyzed, and the results are shown in Table 3 below.

[0107] Table 3

[0108]

[0109] Table 3 shows that the first bunch of bananas had a higher number of seeds, with an average of 0.72 seeds per fruit. The other bunches had a lower number of seeds, with an average of 0-0.06 seeds per fruit.

[0110] In summary, only diploid cultivated banana varieties that can produce mature and fertile pollen can be used as female parents to obtain a high number of seeds. The first bunch of bananas is more likely to produce a high number of seeds. Spraying PGM liquid medium containing 12% glucose immediately after pollination is beneficial to increase the number of seeds and the embryo yield. The hybridization combination of 'Rose Banana' and 'Tropical Small Fruit Banana' combined with embryo in vitro culture has the highest success rate in obtaining hybrid lines.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method of diploid Musa cultivar hybridization, characterized in that, The method comprises the following steps: S1, selecting a rose banana with fertile pollen as a female parent, and selecting a wild banana or a diploid cultivated banana with fertile pollen as a male parent, or selecting a Musa acuminate as a female parent and a Musa acuminata var. siamea as a male parent; S2, artificially crossing the selected female parent and male parent, spraying pollen germination medium after artificial pollination, harvesting hybrid seeds when the fruit is mature; S3, taking the embryo of the hybrid seed for in vitro culture, and transplanting and hardening the rooting seedlings obtained by in vitro culture to obtain hybrid plants. The pollen germination medium comprises 0.01 g / L H3BO3, 0.25 g / L MgSO4·7H2O, 0.25 g / L KNO3, 0.4 g / L Ca(NO3)2, and 12%-18% glucose in water, and has a pH value of 5.

8.

2. The method of claim 1, wherein, The diploid cultivated banana is a cultivated banana with a chromosome ploidy of diploid and a genotype of AA.

3. The method of claim 1, wherein, In S1, the diploid cultivated banana with fertile pollen is selected as the male parent.

4. The method of claim 1, wherein, In S1, the wild banana is selected as the male parent.

5. The method of claim 1 or 4, wherein, The wild banana is a sharp-leaf banana.

6. The method of claim 1, wherein, The artificial pollination is performed in a warm and humid season from March to June or from the middle of August to October. And / or, the artificial pollination is performed as follows: pollen is collected when the anthers of male flowers are mature, split and exposed; and the pollen is applied to the stigma of female flowers when the bracts of female flowers are open and the stigma is exposed. The pollination comb for the artificial pollination is the 1st-3rd comb.

7. The method of claim 1, wherein, The embryo of the hybrid seed is obtained by the following method: after seed disinfection, the seed shell is broken by extrusion, and the embryo is peeled off.

8. The method of claim 1, wherein, The in vitro culture comprises the following steps: the embryo of the hybrid seed is inoculated into an induction medium for dark culture to obtain sprouts, the sprouts are inoculated into a proliferation medium for expansion to obtain clumps, and the clumps are inoculated into a rooting medium for rooting to obtain rooting seedlings.

9. The method of claim 8, wherein, The dark culture is performed for at least one week.

10. The method of claim 8, wherein, The induction medium comprises MS medium, 6-benzylaminopurine 2-3 mg / L, kinetin 1-2 mg / L, adenine 2.5-5 mg / L, agar 4 g / L, and sucrose 30 g / L, and has a pH value of 5.8-6. And / or, the proliferation medium comprises MS medium, 6-benzylaminopurine 2.5-5 mg / L, naphthalene acetic acid 0.1-0.2 mg / L, agar 4 g / L, and sucrose 30 g / L, and has a pH value of 5.8-6. And / or, the rooting medium comprises 1 / 2 MS medium, indole-3-butyric acid 0.5-2 mg / L, naphthalene acetic acid 0.1-0.2 mg / L, 0.5% activated carbon, and agar 4 g / L, and has a pH value of 5.8-6.

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