Crossbreeding method for ludisia discolor

Abstract

The invention discloses a ludisia discolor crossbreeding method which comprises the following steps: spraying 50.0-80.0 mg / L of paclobutrazol in combination with a close planting treatment mode to delay the delayed flowering of an early flowering variety parent plant for 6-12 days, spraying 60.0-90.0 mg / L of 6-BA, adding 28.0-425.0 mg / L of KH2PO4 and 33.0-124.0 mg / L of H3BO33.0 into a matrix, and combining a thin planting treatment mode to promote the late flowering variety parent plant to flower 6-11 days in advance. Or the improved MS culture medium is combined with blue-red composite light to culture ludisia discolor plants in a culture room, the ludisia discolor plants are induced and cultured to bloom, transplanting is not needed, the ludisia discolor plants are directly cultured to bloom, the blooming period is prolonged, and supply of maternal plants in the pollination period is better guaranteed; according to the method, pollination is completed in a time window when ludisia discolor plant male and female parent pollen and stigma activity are optimal and the pollination state is good, the hybrid pollination rate is increased, sterile sowing of hybrid fruit pods is conducted through an improved Kundson C culture medium, germination can be conducted for 28 days, the germination rate is high, seedlings are strong, the development success rate of hybrid embryos is high, and new germplasm is provided for extraction or planting appreciation of special natural drugs.

Description

Technical Field

[0001] The invention relates to the technical field of orchid cultivation, in particular to a method for cross-breeding of Sanguisorba officinalis. Background Art

[0002] Blood orchid belongs to the genus Blood orchid in the family Orchidaceae. The pollination rate is very low under natural conditions, and pollination requires external forces. The pollen activity and stigma activity of Blood orchid plants can last for 1 to 8 days, and the activity drops rapidly after the 9th day. The pollination success rate is very low after more than 12 days. There are large differences in the flowering period of different varieties of Blood orchid, such as Figure 1 As shown, 5-line net pattern red back (left), early flowering period, 3-line red back (right), late flowering period, 5-line green back, late flowering period, such as Figure 2 As shown, at the same time, the variety with 5-line reticulated leaves and light red back has buds, while the variety with 3-line red leaves and back has not yet buds. The flowering period of the two varieties differs by more than 20 days, while the active state of pollen and stigma is maintained for a relatively short time. Therefore, there is a situation where the flowering period does not coincide or the pollination window time is short among the blood leaf orchid varieties, resulting in low pollination success rate, hybridization probability, and low hybrid embryo germination rate.

[0003] At present, the conventional breeding of flowering plants is to plant seedlings or tissue culture seedlings and then transplant them. They will bloom at the end of the year or the beginning of the year. Under artificial conditions, the pollen of the first flowering variety can be collected and stored to pollinate the later flowering variety and serve as the male parent. However, the stigma of the first flowering variety cannot be separated from the plant and stored, and cannot serve as the female parent. Therefore, the number of hybrid combinations that can be configured is limited to a certain extent (i.e., A x B cannot be configured as B x A).

[0004] Due to the differences in flowering periods of varieties, there are few opportunities for pollination and fertilization in the wild or artificial environment, or incomplete fertilization after pollination due to missing the effective period of pollen stigma activity, and poor development of hybrid embryos, which can easily lead to hybridization failure. Therefore, compared with other genera of orchids, there are fewer native species and varieties of blood leaf orchids. For example, there are dozens or hundreds of varieties of golden thread lotus in the genus Alocasia, but there are only a few common blood leaf orchids. Summary of the invention

[0005] The object of the present invention is to provide a method for hybrid breeding of Sanguisorba officinalis to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for hybrid breeding of Sanguisorba officinalis, the method comprising the following steps:

[0007] S1: Select different varieties of blood leaf orchid as parent plants for cultivation, and regulate the flowering period of different varieties of blood leaf orchid plants;

[0008] S2: Prepare reciprocal hybrid combinations, perform artificial pollination, harvest hybrid pods, and sow hybrid pods aseptically using modified Kundson C medium, wherein the formula of the modified Kundson C medium is: Ca(NO 3 ) 2 ·4H 2 O 1000mg / L, MgSO 4 7H 2 O 250mg / L, KH 2 PO 4 280~320mg / L, (NH 4 ) 2 SO 4 680mg / L, KNO 3 600mg / L, FeSO 4 7H 2 O 25.0mg / L, Na 2 EDTA 33.6.1mg / L, MnSO 4 ·4H 2 O 7.5mg / L, glycine 2mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride 0.5mg / L, banana 80g / L, potato 30g / L, peptone 0.8-1.2g / L, sucrose 20g / L, activated carbon 0.6-1.0g / L, the rest is water, pH value is 6.0;

[0009] S3: Sowing hybrid seedlings for transplanting.

[0010] Furthermore, in the improved Kundson C medium, NH 4 + The concentration is 10.29mmol / L.

[0011] Further, the S1 includes:

[0012] A1: Select the blood leaf orchid tissue culture seedlings of relatively uniform size (remove the small seedlings), wash the roots, plant the blood leaf orchid conventional tissue culture propagation seedlings in the substrate, add Huabao No. 2 0.8-1.2g / L, microbial agent 5.0-6.0g / L, spray paclobutrazol 50.0-80.0mg / L, and plant densely to delay the flowering of the early flowering variety parent plants; spray 6-BA 60.0-90.0mg / L, add KH 2 PO 4 238.0~425.0mg / L, H 3 BO 3 93.0~124.0mg / L, and sparse planting, to promote early flowering of late-flowering varieties;

[0013] A2: Place the plants on greenhouse bricks or sand cushion or fence bed for post-transplant management, adjust environmental conditions, keep the ambient temperature no higher than 30℃, and cultivate the plants until they bloom.

[0014] Furthermore, the biological agent includes a Bacillus licheniformis agent.

[0015] Further, the S1 includes:

[0016] B1: Fill the nutrient bag with improved MS culture medium, inoculate 15 to 20 single bud stem segments of Sanguisorba officinalis tissue culture seedlings in each bag of culture medium, the formula of the improved MS culture medium is: (NH 4 ) 2 SO 4 190~220g / L, NH 4 Cl 100~120g / L, Ca(NO 3 ) 2 ·4H 2 O 620g / L, KNO 3 1900~2200g / L, KH 2 PO 3 200~250g / L、Mg(NO 3 ) 2 6H 2 O 380~420g / L, FeSO 4 7H 2 O 27.8mg / L, Na 2 EDTA 37.3mg / L, H 3 BO 3 9.0mg / L, MnSO 4 ·4H 2 O 22.3mg / L, ZnSO 4 7H 2 O 8.6mg / L, KI 0.83mg / L, Na 2 MoO 4 ·2H 2 O 0.25mg / L, glycine 2mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride 0.5mg / L, banana puree 80-100g / L, potato 30-40g / L, peptone 1.0-1.5g / L, sucrose 30g / L, agar powder 5.2-5.6g / L, activated carbon 0.6-1.0g / L, naphthaleneacetic acid 0.5-1.2mg / L, the rest is water, pH value is 5.8;

[0017] B2: After inoculation, place the plants on the culture rack in the culture room and culture them under the conditions of composite light with red and blue light as the main components, light intensity of 4000-5000Lx, and 28℃±1℃ until flower buds appear. Open the bag 15 days after the flower buds appear, let the pedicels stretch out, cover the base of the seedlings with 2 cm thick sterilized substrate, maintain the humidity of the culture medium, open the bag and continue to culture until the cultured plants bloom.

[0018] Furthermore, the material of the matrix is ​​peat: river sand = 2.8-3.6:1 (v / v).

[0019] Furthermore, the spectrum wavelength range of the blue light in the composite light with red and blue light as main components is 380nm to 500nm.

[0020] Furthermore, the spectral wavelength range of the red light in the composite light with red and blue light as main components is 580nm to 780nm.

[0021] Compared with the prior art, the invention has the following beneficial effects: the invention delays the flowering of the early-flowering variety parent plants by 6 to 12 days by spraying 50.0 to 80.0 mg / L of paclobutrazol combined with dense planting, and delays the flowering of the early-flowering variety parent plants by 6 to 12 days by spraying 60.0 to 90.0 mg / L of 6-BA and adding KH 2 PO 4 238.0~425.0mg / L+H 3 BO 3 93.0~124.0mg / L, combined with sparse planting treatment, promote the parent plants of late-flowering varieties to bloom 6~11 days in advance, or use improved MS culture medium combined with blue-red composite light in the culture room to culture blood leaf orchid plants, induce the cultivation of blood leaf orchid plants to bloom, without transplanting blood leaf orchid plants, directly cultivate until flowering, extend its flowering period, better guarantee the supply of mother plants during pollination period, complete pollination in the time window when the pollen and stigma activity of the parents of blood leaf orchid plants is optimal and the pollination state is good, improve the hybrid pollination rate, and use improved Kundson C culture medium for sterile sowing of hybrid fruit pods, which can germinate in 28~35 days, with high germination rate and strong seedlings, improve the success rate of hybrid embryo development, and provide new germplasm for the extraction of special natural medicines or planting and viewing. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are pictures of three varieties of blood leaf orchid in the background technology of the present invention;

[0023] Figure 2 This is a picture showing the difference in flowering period between two varieties of blood leaf orchid in the background technology of the present invention;

[0024] Figure 3 This is a picture of the mutant planted in Example 1 of the present invention;

[0025] Figure 4 This is a diagram of germination conditions of the present invention using Kundson C medium for cultivation;

[0026] Figure 5 This is a diagram of germination conditions of the present invention using improved Kundson C medium for cultivation;

[0027] Figure 6 The spectrum diagram of the composite light with red and blue light as main components of the present invention;

[0028] Figure 7 This is a picture of the bag culture of the present invention;

[0029] Figure 8 This is a picture of the mutant strain transplanted in Example 2 of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Embodiment 1:

[0032] This embodiment provides a method for hybrid breeding of blood leaf orchid, using a 5-line net-patterned light red leaf back variety and a 3-line red leaf back variety as parent plants,

[0033] The seedling tray (50cm×30cm×5cm) was used for transplanting and planting. The seedling tray was divided into three groups. The first group used imported peat: river sand = 3.2:1 (v / v) as the matrix. The blood leaf orchid tissue culture seedlings of relatively uniform size (small seedlings were removed) were selected, the roots were cleaned, and the blood leaf orchid conventional tissue culture propagation seedlings were planted in the matrix. Huabao No. 2 1g / L and microbial agent 5.5g / L were added. The biological agent was selected from Bacillus licheniformis agent. After the matrix was mixed, it was loaded into the seedling tray. Each tray was filled with 4L of matrix, and paclobutrazol 65mg / L was sprayed and densely planted. 150 plants were planted in each tray for dense planting treatment to delay the flowering of the early flowering variety parent plants. 6-BA 75mg / L was sprayed and KH was added to the matrix. 2 PO 4 330mg / L, H 3 BO 3110mg / L, and sparse planting, 90 plants were planted in each tray for sparse planting treatment, to promote early flowering of late flowering varieties of parent plants; the second group used imported peat: river sand = 2.8:1 (v / v) as the matrix, selected blood leaf orchid tissue culture seedlings of relatively uniform size (removing seedlings), washed the roots, planted blood leaf orchid conventional tissue culture propagation seedlings in the matrix, added Huabao No. 2 0.8g / L, microbial agent 5g / L, the biological agent was selected from Bacillus licheniformis agent, the matrix was mixed and loaded into the seedling tray, each tray was filled with 4L matrix, sprayed with paclobutrazol 50mg / L, and dense planting, 150 plants were planted in each tray for dense planting treatment, to delay the flowering of early flowering varieties of parent plants, sprayed with 6-BA 60mg / L, KH 2 PO 4 238mg / L, H 3 BO 3 93mg / L, and sparse planting, 90 plants were planted in each tray for sparse planting treatment, to promote early flowering of late flowering varieties of parent plants; the third group used imported peat: river sand = 3.6:1 (v / v) as the matrix, selected blood leaf orchid tissue culture seedlings of relatively uniform size (removing seedlings), washed the roots, planted blood leaf orchid conventional tissue culture propagation seedlings in the matrix, added Huabao No. 2 1.2g / L, microbial agent 6g / L, the biological agent was selected from Bacillus licheniformis agent, the matrix was mixed and loaded into the seedling tray, each tray was filled with 4L matrix, sprayed with paclobutrazol 80mg / L, and dense planting, 150 plants were planted in each tray for dense planting treatment, to delay the flowering of early flowering varieties of parent plants, sprayed with 6-BA 90mg / L, KH 2 PO 4 425mg / L, H 3 BO 3 124mg / L, and sparse planting, 90 plants per tray in the sparse planting treatment, to promote early flowering of late-flowering varieties of parent plants;

[0034] Place on the greenhouse brick (or sand) cushion or fence bed for post-transplant management. Regulate environmental conditions, with temperature as the main regulating factor, and keep the ambient temperature no higher than 30°C, especially in the hot summer season. The ambient temperature around the transplanted seedlings can be lowered by increasing the ambient humidity and cooling by water evaporation. When cooling down, adjust the light intensity in combination with the double-layer sunshade net to adjust the light in the shed. The light intensity should be 4500Lx. The first group of 5-line net-patterned light red varieties delayed flowering by 6 to 12 days, and 3-line red varieties bloomed 6 to 11 days earlier. The blood leaf orchid period began to show flower buds in mid-December and began to bloom in early January to mid-January. The two varieties can overlap for 12 to 15 days, and the pollination rate is 58%. The second group of 5-line net-patterned light red varieties delayed flowering by 5 to 10 days, and 3-line red varieties bloomed 5 to 10 days earlier. The blood leaf orchid period began in mid-December. Flower buds began to appear in the tenth month, and flowers began to bloom in early January to mid-January. The overlapping days of the pollination period of the two varieties were 9 to 12 days, and the pollination rate was 55%. The plants of the third group of 5-line net-patterned light red leaf varieties bloomed 6 to 11 days later, and the 3-line red leaf varieties bloomed 6 to 11 days earlier. The flower buds began to appear in the mid-December of the blood leaf orchid period, and flowers began to bloom in early January to mid-January. The overlapping days of the pollination period of the two varieties were 10 to 13 days, and the pollination rate was 57%. The experiment showed that the overlapping days of the pollination period of the two varieties in the first group were more, and the pollination rate was higher. The first group was selected for hybridization from the end of January to early February.

[0035] After preparing the reciprocal hybrid group, pollination is completed within 12 days after the first flowering, and the fruit pods are harvested and sown about 45 days later. They need to be harvested in time to prevent the pods from bursting. The appropriate number of hybrid fruit pods is 3 to 5 per plant. The hybrid fruit pods are aseptically sown using the improved Kundson C medium and inoculated into culture bottles. One fruit pod can be sown into 5 to 8 bottles. The formula of the improved Kundson C medium is: Ca(NO 3 ) 2 ·4H 2 O 1000mg / L, MgSO 4 7H 2 O 250mg / L, KH 2 PO 4 300mg / L, (NH 4 ) 2 SO 4 680mg / L, KNO 3 600mg / L, FeSO 4 7H 2 O 25.0mg / L, Na 2 EDTA 33.6.1mg / L, MnSO 4 ·4H 2O 7.5mg / L, glycine 2mg / L, nicotinic acid 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride 0.5mg / L, banana 80g / L, potato 30g / L, peptone 1g / L, sucrose 20g / L, activated carbon 0.8g / L, the rest is water, pH value is 6.0, which is increased in Kundson C medium (NH 4 ) 2 SO 4 180mg / L, making NH 4 + The concentration was increased from 7.56mmol / L to 10.29mmol / L, and KNO 3 600mg / L, KH 2 PO 4 50mg / L; that is, increase the amount of nitrogen, potassium and phosphorus to improve the amount of macronutrients, increase vitamins and organic additives, start germination in 28-35 days, the germination rate is 87%, the protocorm stage is about 22 days, the seedlings grow more evenly, and the seedlings are strong;

[0036] Finally, the hybrid seedlings were transplanted, and the transplanted variants were Figure 3 As shown, three-line plants and five-line reticulate plants germinated from the same bottle of hybrid embryos. The reticulate patterns were different from those of the parental plants. Selecting new germplasm (variety) from the variants can provide new germplasm for the extraction of special natural medicines or planting for viewing.

[0037] Comparative Example 1:

[0038] Similarly, the 5-line net-patterned light red leaf variety and the 3-line red leaf variety were used as parent plants and transplanted in seedling trays (50cm×30cm×5cm) and placed on greenhouse brick (or sand) cushions or on fence beds for post-transplant management. At the same time, the 5-line net-patterned light red leaf variety had buds, while the 3-line red leaf variety had not yet budded. The flowering period difference between the two varieties was more than 20 days, the number of days of overlapping pollinable flowering periods was 0, there were no pollinable plants, and there were no hybrid fruit pods.

[0039] Comparative Example 2:

[0040] Similarly, the 5-line net-patterned light red on the back variety and the 3-line red on the back variety were used as parent plants, and transplanted and planted in a seedling tray (50cm×30cm×5cm) matrix, with imported peat: river sand = 3.2:1 (v / v) as the matrix, and the blood leaf orchid tissue culture seedlings of relatively uniform size (removing the seedlings) were selected, the roots were washed, and the blood leaf orchid conventional tissue culture propagation seedlings were planted in the matrix, and Huabao No. 2 1g / L and microbial agent 5.5g / L were added. The biological agent was selected from Bacillus licheniformis agent. After the matrix was mixed, it was loaded into the seedling tray, each tray was filled with 4L of matrix, and dense planting was performed. 150 plants were planted in each tray for dense planting treatment. In early December (about 30 days before flowering, when the plant exposed the sword tongue leaf (i.e., the last incomplete leaf)), 65mg / L of paclobutrazol was sprayed to delay the flowering of the early flowering variety parent plants; 75mg / L of 6-BA was sprayed, and KH was added to the matrix. 2 PO 4 330mg / L, H 3 BO 3 110mg / L, and sparse planting, 90 plants per tray in the sparse planting treatment, to promote early flowering of late-flowering varieties of parent plants;

[0041] Place on the greenhouse brick (or sand) cushion or fence bed for post-transplant management. Regulate environmental conditions, with temperature as the main regulating factor, and keep the ambient temperature no higher than 30°C, especially in the hot summer season. The ambient temperature around the transplanted seedlings can be lowered by increasing the ambient humidity and cooling by water evaporation. When cooling down, adjust the light intensity in combination with a double-layer sunshade net to adjust the light. The light intensity should be 4500Lx. The 5-line net-patterned light red back variety will delay flowering by 6 to 12 days, and the 3-line red back variety will bloom 6 to 11 days earlier. The flower buds of the blood leaf orchid begin to appear in mid-December, and the flowers begin to bloom from early to mid-January. The two varieties can be pollinated and the overlapping days of flowering are 12 to 15 days, and the pollinated plant rate is 58%. Hybridization is carried out from the end of January to early February.

[0042] After preparing the reciprocal hybrid group, pollination is completed within 12 days after the first flowering. The fruit pods are harvested and sown about 45 days later. They need to be harvested in time to prevent the pods from bursting. The appropriate number of hybrid fruit pods is 3 to 5 per plant. The hybrid fruit pods are aseptically sown through the basic Kundson C medium and inoculated into the culture bottle. One fruit pod can be sown into 5 to 8 bottles. The basic Kundson C medium formula is as follows: Ca(NO 3 ) 2 ·4H 2 O 1000mg / L, MgSO 4 7H 2 O 250mg / L, KH 2 PO 4 250mg / L, (NH 4 ) 2 SO 4500mg / L, FeSO 4 7H 2 O 25.0mg / L, Na 2 EDTA 33.6.1mg / L, MnSO 4 ·4H 2 O 7.5mg / L, sucrose 20g / L, agar 15mg / L, the rest is water, pH value is 6.0; it starts to germinate in 50-60 days, the germination rate is 46%, the protocorm stage is about 28 days, the seedlings grow more evenly, and the seedlings are stronger. Finally, the hybrid seedlings are sown for transplanting. After transplanting, a small number of mutants appear.

[0043] As shown in the following table 1, the pollination conditions of different varieties of blood leaf orchid under different flowering period treatments under substrate cultivation conditions and the differences in hybrid seed cultivation under different culture media are shown.

[0044] Table 1

[0045]

[0046]

[0047] As shown in Table 1, the hybrid pollination rate was significantly increased after the flowering period was adjusted. Figure 4 and Figure 5 As shown, the germination rate of hybrid embryos was low when the basic Kundson C medium was used for culture, while the germination rate of hybrid embryos was high when the improved Kundson C medium was used for culture.

[0048] Embodiment 2:

[0049] This embodiment provides a method for hybrid breeding of blood leaf orchid, which also uses a 5-line net-patterned light red leaf back variety and a 3-line red leaf back variety as parent plants, and directly cultivates them until flowering using a modified MS culture medium and composite light with red and blue light as main components, without transplanting the blood leaf orchid plants;

[0050] Specifically, a culture bag (bottle) is filled with a modified MS culture medium, and each bag of culture medium is inoculated with a single bud stem segment of a tissue culture plant of a blood leaf orchid. The formula of the modified culture medium is: (NH 4 ) 2 SO 4 210g / L, NH 4 Cl 110g / L, Ca(NO 3 ) 2 ·4H 2 O620g / L, KNO 3 2000g / L, KH 2 PO 3 230g / L、Mg(NO 3 )2 6H 2 O 400g / L, FeSO 4 7H 2 O 27.8mg / L, Na 2 EDTA 37.3mg / L, H 3 BO 3 9.0mg / L, MnSO 4 ·4H 2 O 22.3mg / L, ZnSO 4 7H 2 O 8.6mg / L, KI 0.83mg / L, Na 2 MoO 4 ·2H 2 O 0.25mg / L, glycine 2mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride 0.5mg / L, banana puree 90g / L, potato 35g / L, peptone 1.2g / L, sucrose 30g / L, agar powder 5.4g / L, activated carbon 0.8g / L, naphthaleneacetic acid 0.8mg / L, and the rest is water. The pH value is 5.8. The modified MS medium adopts (NH 4 ) 2 SO 4 NH 4 Cl and Ca(NO 3 ) 2 Substitute NH in ammonium nitrate of MS medium 4 + -N and NO 3 -N, add banana puree, potato, and peptone to provide the nutrient requirements of blood leaf orchid seedlings, no need to add inositol, save costs, and increase KNO 3 100mg / L, KH 2 PO 3 60g / L, H 3 BO 3 2.8mg / L, P element promotes plant reproductive growth, B element promotes plant flower organ morphology and development, increasing the amount of P and B elements promotes the vegetative growth of blood leaf orchid plants and better transition to reproductive growth and flower organ formation and development in the later stage; PP bag culture medium, each bag contains 250ml culture medium, 0.12MPa sterilization for 22min. Inoculate in early April, inoculate 15 to 20 blood leaf orchid tissue culture plant single bud stem segments per bag of culture medium, such as Figure 6 As shown,

[0051] After inoculation, it is placed on the culture rack in the culture room. In the composite light with red and blue light as the main components, the spectral wavelength range of blue light in the composite light with red and blue light as the main components is 380nm~500nm, accounting for about 30.0% of the spectrum, and the spectral wavelength range of red light in the composite light with red and blue light as the main components is 580nm~780nm, accounting for about 45.0% of the spectrum. Its spectrum is as shown in Figure 6 As shown;

[0052] The seeds were cultured under light intensity of 4500Lx and 28℃±1℃. Flower buds began to appear after about 250 days of culture (mid-December). The bags were opened 15 days after the appearance of flower buds (to allow the pedicels to stretch out). The base of the seedlings was covered with a 2 cm thick sterilized peat: river sand = 3.2:1 (v / v) matrix (to maintain the humidity of the culture medium). The bags were opened and culture was continued until the cultured plants bloomed. Flowers began to bloom in early to mid-January. The two varieties had overlapping days of pollination for 12 to 15 days, and the rate of pollinable plants was 56%. Hybridization was carried out from the end of January to early February.

[0053] After preparing the reciprocal hybrid group, pollination is completed within 12 days after the first flowering, and the fruit pods are harvested and sown about 45 days later. They need to be harvested in time to prevent the pods from bursting. The suitable number of hybrid fruit pods is 3 to 5 per plant. The hybrid fruit pods are aseptically sown through the improved Kundson C medium and planted in culture bags. One fruit pod can be sown in 5 to 8 bags. The formula of the improved Kundson C medium is: Ca(NO 3 ) 2 ·4H 2 O 1000mg / L, MgSO 4 7H 2 O 250mg / L, KH 2 PO 4 300mg / L, (NH 4 ) 2 SO 4 680mg / L, KNO 3 600mg / L, FeSO 4 7H 2 O 25.0mg / L, Na 2 EDTA 33.6.1mg / L, MnSO 4 ·4H 2 O 7.5mg / L, glycine 2mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride 0.5mg / L, banana 80g / L, potato 30g / L, peptone 1.0g / L, sucrose 20g / L, activated carbon 0.8g / L, the rest is water, pH value is 6.0, modified Kundson C medium is based on Kundson C medium with (NH 4 ) 2 SO4 180mg / L, making NH 4 + The concentration was increased from 7.56mmol / L to 10.29mmol / L, and KNO 3 600mg / L, KH 2 PO 4 50mg / L; that is, increase the amount of nitrogen and potassium to improve the amount of macronutrients, increase vitamins and organic additives, start germination in 28-35 days, the germination rate is 87%, the protocorm stage is about 22 days, the seedlings grow more evenly, and the seedlings are strong;

[0054] Finally, the hybrid seedlings were transplanted, and the transplanted variants were Figure 7 As shown, three-line plants and five-line reticulate plants germinated from the same bag of hybrid embryos. The reticulate patterns were different from those of the parental plants. Selecting new germplasm (variety) from the variants can provide new germplasm for the extraction of special natural medicines or planting for viewing.

[0055] Comparative Example 3:

[0056] Similarly, the 5-line net-patterned light red leaf back variety and the 3-line red leaf back variety were used as parent plants, and the nutrient bags were filled with MS culture medium. Each bag of culture medium was inoculated with a single bud stem segment of a blood leaf orchid tissue culture seedling plant. The basic culture medium formula was as follows: NH 4 NO 3 1650g / L, KNO 3 1900g / L, KH 2 PO 3 170g / L, MgSO 4 7H 2 O 370g / L, CaCl 2 440mg / L, KI0.83mg / L, H 3 BO 3 9.0mg / L, MnSO 4 ·4H 2 O 22.3mg / L, ZnSO 4 7H 2 O 8.6mg / L, Na 2 MoO 4 ·2H 2 O0.25mg / L, CuSO 4 ·5H 2 O 0.025mg / L, CoCl 2 ·5H 2 O 0.025mg / L, FeSO 4 7H 2 O 27.8mg / L, Na 2EDTA 37.3mg / L, inositol 100mg / L, glycine 2mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride 0.5mg / L, sucrose 30g / L, agar 6g / L, the rest is water, pH value is 5.8;

[0057] Use PP bags to pack the culture medium, each bag contains 250 ml of culture medium, sterilize at 0.12 MPa for 22 minutes. Inoculate in early April, inoculate 15 to 20 single bud stem segments of Sanguisorba officinalis tissue culture seedlings in each bag of culture medium;

[0058] After inoculation, place them on the culture rack in the culture room and culture them under normal light conditions at 28℃±1℃ for about 250 days (mid-December). The 5-line reticulated leaf back light red variety only grows dragon roots (reproductive rhizomes), no leaves, and no flowers. The 3-line red leaf back variety grows dragon roots accompanied by a small number of seedlings with leaves. The seedlings do not bloom and cannot be prepared into hybrid combinations for hybridization.

[0059] Comparative Example 4:

[0060] Similarly, the 5-line net-patterned light red leaf back variety and the 3-line red leaf back variety were used as parent plants, and the MS medium was filled with nutrients, and each bag of medium was inoculated with a single bud stem segment of the blood leaf orchid tissue culture seedling plant. The MS medium was the same as in comparative example 3, and the medium was packed in PP bags, each bag was filled with 250 ml of medium, and sterilized at 0.12 MPa for 22 minutes. Inoculation was carried out in early April, and 15 to 20 single bud stem segments of the blood leaf orchid tissue culture seedling plant were inoculated in each bag of medium;

[0061] After inoculation, the seeds were placed on the culture rack in the culture room and cultured under the conditions of composite light with red and blue light as the main components, light intensity of 4500Lx, and 28℃±1℃. After about 250 days of culture (mid-December), the 5-line reticulated leaf back light red variety grew dragon roots accompanied by a small number of leaf seedlings, and the seedlings did not bloom; the 3-line leaf back red variety grew a small number of dragon roots accompanied by a small number of leaf seedlings, and the seedlings did not bloom, and hybrid combinations could not be prepared for hybridization.

[0062] Comparative Example 5:

[0063] Similarly, the 5-line net-patterned light red leaf back variety and the 3-line red leaf back variety were used as parent plants, and the modified MS medium was filled with nutrients, and each bag of the medium was inoculated with a single bud stem segment of the blood leaf orchid tissue culture seedling plant. The formula of the modified MS medium was as in Example 2, and the medium was packed in PP bags, each bag was filled with 250 ml of the medium, and sterilized at 0.12 MPa for 22 minutes. Inoculation was carried out in early April, and 15 to 20 single bud stem segments of the blood leaf orchid tissue culture seedling plant were inoculated in each bag of the medium;

[0064] After inoculation, the plants were placed on a culture rack in a culture room and cultured under normal light conditions at 28°C ± 1°C. Flower buds began to appear after about 250 days of culture (mid-December). The bags were opened 15 days after the flower buds appeared (to allow the pedicels to stretch out), and the base of the seedlings was covered with a 2 cm thick sterilized peat: river sand = 2.8-3.6:1 (v / v) matrix (to maintain the humidity of the culture medium). The bags were opened and continued to be cultured until the cultured plants bloomed. At the same time, the 5-line net-patterned light red variety on the back of the leaves had buds, and the 3-line red variety on the back of the leaves had not yet appeared flower buds. The difference in flowering period between the two varieties was 16-20 days. The overlapping days of the pollination flowering period of the two varieties were 2-3 days, and the pollination plant rate was 5%. Hybridization was carried out from the end of January to the beginning of February.

[0065] After preparing the reciprocal hybridization group, pollination is completed within 12 days after the initial flowering, and the fruit pods are harvested and sown about 45 days later, and the fruit pods need to be harvested in time to prevent the pods from bursting. The number of suitable hybrid fruit pods is 1-2 per plant, and the hybrid fruit pods are aseptically sown through the basic Kundson C medium. The basic Kundson C medium is as in comparative example 2, and the germination begins at 56 days, the germination rate is 23%, the protocorm stage is about 45 days, and the growth of the seedlings is relatively uniform. Finally, the hybrid seedlings are transplanted and planted, and after the transplanting and planting, the probability of selecting a variant is low.

[0066] Comparative Example 6:

[0067] Similarly, the 5-line net-patterned light red leaf back variety and the 3-line red leaf back variety were used as parent plants, and the modified MS medium was filled with nutrients, and each bag of the medium was inoculated with a single bud stem segment of the blood leaf orchid tissue culture seedling plant. The formula of the modified MS medium was as in Example 2, and the medium was packed in PP bags, each bag was filled with 250 ml of the medium, and sterilized at 0.12 MPa for 22 minutes. Inoculation was carried out in early April, and 15 to 20 single bud stem segments of the blood leaf orchid tissue culture seedling plant were inoculated in each bag of the medium;

[0068] After inoculation, the plants were placed on a culture rack in a culture room and cultured under normal light conditions at 28°C ± 1°C. Flower buds began to appear after about 250 days of culture (mid-December). The bags were opened 15 days after the flower buds appeared (to allow the pedicels to stretch out), and the base of the seedlings was covered with a 2 cm thick sterilized peat: river sand = 2.8-3.6:1 (v / v) matrix (to maintain the humidity of the culture medium). The bags were opened and cultured until the plants bloomed. At the same time, the 5-line net-patterned light red variety on the back of the leaves had buds, and the 3-line red variety on the back of the leaves had not yet appeared flower buds. The difference in flowering period between the two varieties was 16-20 days. The overlapping days of the pollination flowering period of the two varieties were 2-3 days, and the pollination plant rate was 5%. Hybridization was carried out from the end of January to the beginning of February.

[0069] After preparing the reciprocal hybridization group, pollination is completed within 12 days after the initial flowering, and the fruit pods are harvested and sown about 45 days later, and the pods need to be harvested in time to prevent the pods from bursting. The suitable number of hybrid fruit pods is 1-2 per plant, and the hybrid fruit pods are aseptically sown through the basic Kundson C medium. The basic Kundson C medium is as in comparative example 2, and the germination begins in 28 days, the germination rate is 85%, the protocorm stage is 28 days, and the seedlings grow more neatly and strongly. Finally, the sowing hybrid seedlings are transplanted and planted, and after the transplanting and planting, variant strains are selected.

[0070] Comparative Example 7:

[0071] Similarly, the 5-line net-patterned light red leaf back variety and the 3-line red leaf back variety were used as parent plants, and the modified MS medium was filled with nutrients, and each bag of the medium was inoculated with a single bud stem segment of the blood leaf orchid tissue culture seedling plant. The formula of the modified MS medium was as in Example 2, and the medium was packed in PP bags, each bag was filled with 250 ml of the medium, and sterilized at 0.12 MPa for 22 minutes. Inoculation was carried out in early April, and 15 to 20 single bud stem segments of the blood leaf orchid tissue culture seedling plant were inoculated in each bag of the medium;

[0072] After inoculation, the plants were placed on a culture rack in a culture room and cultured under a composite light with red and blue light as the main components, a light intensity of 4500Lx, and 28°C±1°C. Flower buds began to appear after about 250 days of culture (mid-December). The bags were opened 15 days after the flower buds appeared (to allow the pedicels to stretch out), and the base of the seedlings was covered with a 2 cm thick sterilized peat: river sand = 2.8-3.6:1 (v / v) matrix (to maintain the humidity of the culture medium). The bags were opened and cultured until the cultured plants bloomed. The overlapping days of the pollination flowering period of the two varieties were 12-15 days, and the pollination plant rate was 56%. Hybridization was carried out from the end of January to the beginning of February.

[0073] After preparing the reciprocal hybridization group, pollination is completed within 12 days after the initial flowering, and the fruit pods are harvested and sown about 45 days later, and the fruit pods need to be harvested in time to prevent the pods from exploding. The number of suitable hybrid fruit pods is 3-5 per plant, and the hybrid fruit pods are aseptically sown through the basic Kundson C medium. The basic Kundson C medium is as in comparative example 2, and the germination begins at 42 days, the germination rate is 47%, the protocorm stage is about 34 days, and the growth of the seedlings is more uniform and stronger. Finally, the hybrid seedlings are transplanted and planted, and after the transplanting and planting, the mutation is selected.

[0074] As shown in the following table, Table 2 shows the pollination conditions of different blood leaf orchid varieties under different flowering period treatments under substrate cultivation conditions and the differences in hybrid seed cultivation under different culture media.

[0075] Table 2

[0076]

[0077] As shown in Table 2, after adjusting the flowering period, the hybrid pollination rate was significantly improved. Figure 4 and Figure 5 As shown, the germination rate of hybrid embryos was low when the basic Kundson C medium was used for culture, while the germination rate of hybrid embryos was high when the improved Kundson C medium was used for culture.

[0078] The invention delays the flowering of the early flowering variety parent plants by 6 to 12 days by spraying 50.0 to 80.0 mg / L of paclobutrazol combined with dense planting, spraying 6-BA 60.0 to 90.0 mg / L, and adding KH 2 PO 4 238.0~425.0mg / L+H 3 BO 3 93.0~124.0mg / L, combined with sparse planting treatment, promote the parent plants of late-flowering varieties to bloom 6~11 days in advance, or use improved MS culture medium combined with blue-red composite light in the culture room to culture blood leaf orchid plants, induce the cultivation of blood leaf orchid plants to bloom, without transplanting blood leaf orchid plants, directly cultivate until flowering, extend its flowering period, better guarantee the supply of mother plants during pollination period, complete pollination in the time window when the pollen and stigma activity of the parents of blood leaf orchid plants is optimal and the pollination state is good, improve the hybrid pollination rate, and use improved Kundson C culture medium for sterile sowing of hybrid fruit pods, which can germinate in 28~35 days, with high germination rate and strong seedlings, improve the success rate of hybrid embryo development, and provide new germplasm for the extraction of special natural medicines or planting and viewing.

[0079] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for crossbreeding of Sanguisorba officinalis, characterized in that: The method comprises the following steps: S1: Select different varieties of blood leaf orchid as parent plants for cultivation, and regulate the flowering period of different varieties of blood leaf orchid plants; S2: Prepare reciprocal hybrid combinations, perform artificial pollination, harvest hybrid pods, and sow hybrid pods aseptically using modified Kundson C medium, wherein the formula of the modified Kundson C medium is: Ca(NO3)2·4H2O 1000mg / L, MgSO4·7H2O 250mg / L, KH2PO4 280-320mg / L, (NH4)2SO4 680mg / L, KNO3 600mg / L, FeSO4·7H2O 25.0mg / L, Na2EDTA 33.6.1mg / L, MnSO4·4H2O 7.5mg / L, glycine 2mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride 0.5mg / L, banana 80g / L, potato 30g / L, peptone 0.8-1.2g / L, sucrose 20g / L, activated carbon 0.6-1.0g / L, the rest is water, pH value is 6.0; S3: Sowing hybrid seedlings for transplanting.

2. The method for hybrid breeding of Sanguisorba officinalis according to claim 1, characterized in that: In the modified Kundson C medium, NH4 + The concentration is 10.29mmol / L.

3. The method for hybrid breeding of Sanguisorba officinalis according to claim 1, characterized in that: The S1 includes: A1: Select the blood leaf orchid tissue culture seedlings of relatively uniform size (remove the small seedlings), wash the roots, plant the blood leaf orchid conventional tissue culture propagation seedlings in the substrate, add 0.8-1.2g / L of Huabao No. 2, 5.0-6.0g / L of microbial agent, spray 50.0-80.0mg / L of paclobutrazol, and plant densely to delay the flowering of the early-flowering parent plants; spray 6-BA 60.0-90.0mg / L, add KH2PO4 238.0-425.0mg / L and H3BO3 93.0-124.0mg / L to the substrate, and plant sparsely to promote the early flowering of the late-flowering parent plants; A2: Place the plants on greenhouse bricks or sand cushion or fence bed for post-transplant management, adjust environmental conditions, keep the ambient temperature no higher than 30℃, and cultivate the plants until they bloom.

4. The method for hybrid breeding of Sanguisorba officinalis according to claim 3, characterized in that: The biological agent includes a Bacillus licheniformis agent.

5. The method for hybrid breeding of Sanguisorba officinalis according to claim 1, characterized in that: The S1 includes: B1: Fill the nutrient bag with improved MS culture medium, inoculate 15-20 single bud stem segments of Sanguisorba officinalis tissue culture seedlings in each bag of culture medium, the formula of the improved MS culture medium is: (NH4)2SO4 190-220g / L, NH4Cl 100-120g / L, Ca(NO3)2·4H2O 620g / L, KNO3 1900-2200g / L, KH2PO3 200-250g / L, Mg(NO3)2·6H2O 380-420g / L, FeSO4·7H2O 27.8mg / L, Na2EDTA 37.3mg / L, H3BO3 9.0mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, KI 0.83mg / L, Na2MoO4·2H2O 0.25mg / L, glycine 2mg / L, niacin 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride 0.5mg / L, banana puree 80-100g / L, potato 30-40g / L, peptone 1.0-1.5g / L, sucrose 30g / L, agar powder 5.2-5.6g / L, activated carbon 0.6-1.0g / L, naphthaleneacetic acid 0.5-1.2mg / L, the rest is water, pH value is 5.8; B2: After inoculation, place the plants on the culture rack in the culture room and culture them under the conditions of composite light with red and blue light as the main components, light intensity of 4000-5000Lx, and 28℃±1℃ until flower buds appear. Open the bag 15 days after the flower buds appear, let the pedicels stretch out, cover the base of the seedlings with 2 cm thick sterilized substrate, maintain the humidity of the culture medium, open the bag and continue to culture until the cultured plants bloom.

6. The method for hybrid breeding of Sanguisorba officinalis according to claim 3 or 6, characterized in that: The material of the matrix is ​​peat: river sand = 2.8-3.6:1 (v / v).

7. The method for hybrid breeding of Sanguisorba officinalis according to claim 1, characterized in that: The spectrum wavelength range of the blue light in the composite light with red and blue light as main components is 380nm to 500nm.

8. The method for hybrid breeding of Sanguisorba officinalis according to claim 1, characterized in that: The spectral wavelength range of the red light in the composite light with red and blue light as main components is 580nm to 780nm.

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