CN101116423A - A kind of Primulina tabacum Hance tissue culture propagation and field cultivation method - Google Patents

Abstract

A method for tissue culture propagation and field cultivation of Primulina tabacum Hance. The present invention uses Primulina tabacum Hance plant leaves collected in the field as explants. For the cultivation stages such as transplanting steps and field transplanting steps, the medium formulations of various cultivation stages such as induction, propagation, rooting, and transplanting are allocated and optimized, so that the primula moss can realize the purpose of its effective propagation. Combining with the principle of ecological restoration, the test-tube seedlings are planted in the locality where Primula is found, so as to realize the planting survival and ecological restoration of Primula in the original place.

Description

A kind of Primulina tabacum Hance tissue culture propagation and field cultivation method

technical field

The present invention relates to a kind of biological technology for in vitro preservation and breeding of rare and endangered plant Primula tabacum Hance, in particular, relates to a kind of tissue culture breeding and breeding of Primulina tabacum Hance. Field cultivation method.

Background technique

Primulina tabacum Hance (Primulina tabacum Hance) is a small perennial herb of Gesneriaceae. Since American Henry discovered the rare plant Primulina tabacum Hance on the stone wall of the Lianjiang River Basin in Lianzhou, northern Guangdong in 1881, it has been The mystery disappeared for 120 years. Until a few years ago, relevant domestic media reported that Primula moss, the first batch of first-class endangered plants in the country, was discovered in Lianzhou. Its discovery is undoubtedly of great academic value to the study of Lingnan paleoclimate, soil and animal and plant evolution. The use of plant tissue culture technology to carry out plant protection and propagation has been successful in many plant species. According to different types of plants, there are great differences in the cultivation conditions and methods required for in vitro cultivation. As the first batch of rare and endangered plants under the key first-level protection in China, Primula laxatives, due to the requirements of the living environment of this species Special, narrow geographical distribution, small population. At present, the use of plant biotechnology to propagate and cultivate Primula moss, to protect and utilize this rare and endangered plant, has not been reported at home and abroad.

Contents of the invention

In order to protect Primulina tabacum Hance, a rare and endangered plant, the applicant carried out research work on plant tissue culture, successfully induced somatic embryogenesis and the formation of adventitious buds, and thus established effective reproductive System and plant regeneration system, combined with the principle of restoring ecology to realize the planting and survival of primula moss in the original place, thus the present invention is proposed.

The purpose of this invention is to propose a kind of Primulina tabacum Hance tissue culture propagation and field cultivation method.

Primulina tabacum Hance tissue culture propagation and field cultivation method proposed by the present invention, its technical scheme is as follows: use the Primulina tabacum Hance plant blade collected in the field as an explant, and carry out cultivation propagation and cultivation according to the following steps :

(1) Induction culture step: the leaf explants of Primula moss plant are inserted into the induction medium for culture, and the somatic embryogenesis or/and adventitious bud formation of the Primula moss leaf explants are induced. Cultivate in the dark at ℃ or irradiate with low light at 1-20 μmolm ^-2 s ^-1 for 12-14 hours per day, and cultivate for 50-70 days. After the explants can induce the formation of somatic embryos or adventitious buds, continue to cultivate for 20-30 day, the induction medium is based on MS basic medium, supplemented with calcium 220-440mg L ^-1 , sucrose 20-30g L ^-1 , pH value 6.5-7.5, and NAA 0.5-1.0mg L ^{-1 1} +TDZ 0.5-1.0 mg L ^-1 , or BAP 0.5-1.0 mg L ^-1 +TDZ 0.2-1.0 mg L ^-1 , or BAP 0.5-1.0 mg L ^-1 +NAA 0.5-1.0 mg L ^-1 . Among them, NAA 0.5～1.0mg L ^-1 +TDZ 0.5～1.0mg L ^-1 can induce somatic embryogenesis; BAP0.5～1.0mg L ^-1 +NAA 0.5～1.0mg L ^-1 can induce the formation of adventitious buds; BAP 0.5～1.0mg L ^-1 + TDZ 0.2～1.0mg L ^-1 can induce the formation of somatic embryos and adventitious buds at the same time. On average, each explant can induce about 80-90 somatic embryos or adventitious buds.

(2) Cluster bud propagation step: transfer somatic embryos or adventitious buds to the propagation medium for culture, the conditions are 20-30°C, 20-50 μmol m ^-2 s ^-1 light per day for 14 hours, one month to Subculture once every one and a half months, and the breeding frequency can reach 5-8. The breeding medium is based on MS basic medium, 220-440 mg L ^-1 of calcium, 20-30 g L ^{-1 of} sucrose, and pH 6.5-7.5. , plus BAP 0.5～1.0mg L ^-1 +NAA 0.1～0.5mg L ^-1 ;

(3) Steps of rooting culture: Cut the cluster buds and transfer them to the rooting medium for light culture to induce rooting to become ^{test -} tube plantlets. The medium is based on MS basic medium, supplemented with calcium 220-440mg L ^-1 , sucrose 20-30g L ^-1 , pH value 6.5-7.5, and IBA 0.2-1.0mgL ^-1 or/and 0.2% activity carbon, the test-tube plantlets were transplanted after being cultivated on the medium for 1 month;

(4) Transplanting step: the test-tube seedling of long root is transplanted in the matrix that is transplanted into sand basin, is placed in the cultivation under the environment of moistening, shade, and the matrix in sand basin presses limestone: vermiculite: sandstone=( 1～2):(2～4):(1～2) ratio configuration;

(5) Field transplanting step: plant the test-tube seedlings after the strong seedlings in the above-mentioned sand basins in the limestone cave environment, and plant together with the associated species moss.

In the above-mentioned induction culture step, the preferred formula of the induction medium is: based on MS basic medium, 440 mg L ^-1 of calcium supplemented, 20-30 g L ^-1 of sucrose, pH value of 6.5-7.0, and additional Add NAA 0.5mg L ^-1 + TDZ 1.0mg L ^-1 , or BAP 0.5mg L ^-1 + TDZ 0.2mg L ^-1 , or BAP 1.0mg L ^-1 + NAA 0.5mg L ^-1 ;

In the above-mentioned cluster bud propagation step, the preferred formulation of the propagation medium is: based on MS basic medium, 440 mg L ^-1 of calcium supplemented, 20-30 g L ^-1 of sucrose, and a pH value of 6.5-7.0. Added BAP 1.0mg L ^-1 +NAA 0.1mg L ^-1 .

In the above-mentioned rooting culture step, the preferred formulation of the rooting medium is: based on MS basic medium, 440 mg L ^-1 of calcium supplemented, 20-30 g L ^-1 of sucrose, pH 6.5-7.0, and additional Added IBA 1.0mg L ^-1 or/and 0.2% activated carbon.

In the above-mentioned transplanting steps, it is better to configure the matrix in the sand basin where the test-tube seedlings are transplanted according to the ratio of limestone: vermiculite: sandstone=1:2:1.

The invention provides a series of suitable formulas and culture conditions, including formulas for a series of basic processes such as induction, propagation, rooting, transplanting, etc., so that the primula moss can realize the purpose of effective propagation. Combining with the principle of ecological restoration, the test-tube seedlings are planted in the locality where Primula is found, so as to realize the planting survival and ecological restoration of Primula in the original place.

a) Induction culture stage: by adjusting the formula of the growth regulator, the somatic embryogenesis or the formation of adventitious buds in the leaf explants of Primula moss can be induced purposefully;

b) Cluster bud propagation stage: Transferring somatic embryos or adventitious buds to the propagation medium can establish a cluster bud propagation system, and screen the optimum medium to obtain a higher proliferation frequency;

c) rooting stage: the cluster buds are cut and transferred to the screened optimal rooting medium for light culture to induce rooting, and the test-tube plantlets can take root within 1 month of cultivation on the medium;

d) Transplanting stage: the test-tube seedlings with long roots are transplanted outdoors, and the substrates of limestone, vermiculite and sandstone are utilized and additionally placed in a moist, shady environment for cultivation, so as to produce adaptation to Primula moss liking high calcium and The growth environment with high pH value ensures that the test-tube seedlings have a high transplanting survival rate, and the transplanting survival rate reaches 85-90%;

e) Field transplanting test: Using the principle of species regression in restoration ecology, recreating and restoring habitats and improving the survival rate; through the application of this project, the total survival rate is as high as 80%, which can enable the preservation and development of this national first-class endangered plant , which is beneficial to the vegetation restoration of cave entrances in limestone areas, and lays a good foundation for further utilization of this species in the future.

Detailed ways

Embodiment one

Using the leaves of Primulina tabacum Hance collected from the Lianzhou underground river scenic spot as explants, the tissue culture propagation and field cultivation of Primulina tabacum Hance are carried out, and the specific steps are as follows:

(1) Induction culture step: graft the leaf explants of the Primula moss plant into the induction medium for culture, induce the somatic embryogenesis and the formation of adventitious buds of the Primula moss leaf explants, and the induction medium is based on MS Medium, supplemented with calcium 440mgL ^-1 , sucrose 30g L ^-1 , based on pH 7.0, additionally added BAP 0.5mg L ^-1 +TDZ 0.2mg L ^-1 , culture conditions: 20～30℃, 10μmol per day m ^-2 s ^-1 was irradiated with weak light for 14 hours, and cultured for 60 days. After the explants could induce the formation of somatic embryos and adventitious buds, they continued to culture for 30 days. On average, each explant can induce about 80-90 somatic embryos or adventitious buds.

(2) Cluster bud propagation step: transfer somatic embryos or adventitious buds to the propagation medium for culture, the conditions are 20-30°C, 30 μmol m ^-2 s ^-1 light per day for 14 hours, subculture once a month , the propagation medium is based on MS basic medium, supplemented with calcium 440mg L ^-1 , sucrose 30g L ^-1 , pH 6.5, and additionally added BAP 1.0mg L ^-1 +NAA 0.1mgL ^-1 ;

(3) Rooting culture step: cut the cluster buds and transfer ^them to the rooting medium for light culture to induce rooting to become test ^- tube plantlets. Based on MS basic medium, supplemented with calcium 440mg L ^-1 , sucrose 20g L ^-1 , pH value 6.5, additionally added IBA 1.0mg L ^-1 and 0.2% activated carbon. The test-tube seedlings were transplanted after being cultured on the medium for 1 month;

(4) Transplanting steps: transplant the test-tube seedlings with long roots into the substrate transplanted into the sand basin, and place them in a moist, shady environment to cultivate. The substrate in the sand basin is based on limestone: vermiculite: sandstone=1 : 2:1 ratio configuration;

(5) Field transplanting step: plant the test-tube seedlings after the strong seedlings in the above-mentioned sand basins in the limestone cave environment, and plant together with the associated species moss.

Embodiment two

Using the leaves of Primulina tabacum Hance collected from the Lianzhou underground river scenic spot as explants, the tissue culture propagation and field cultivation of Primulina tabacum Hance are carried out, and the specific steps are as follows:

(1) Induction culture step: the leaf explants of Primula moss plant are inserted into the induction medium for culture, and the somatic embryogenesis of the leaf explants of Primula moss is induced, and the induction medium is MS basic medium, with additional calcium 400mg L ^-1 , 30g L ^-1 sucrose, based on pH 6.5, plus NAA 0.5mg L ^-1 +TDZ 1.0mg L ^-1 , culture conditions: 20～30℃, 5μmol m ^-2 s per day ^-1 Irradiate with weak light for 14 hours, culture for 60 days, and continue to culture for 30 days after the explants can be induced to form somatic embryos.

(2) Cluster bud propagation step: transfer the somatic embryos to the propagation medium for culture under the conditions of 20-30° C., 20 μmol m ^-2 s ^-1 light per day for 14 hours, and subculture once a month. The propagation medium is based on MS basic medium, supplemented with calcium 400mg L ^-1 , sucrose 30g L ^-1 , pH 7.0, and BAP 1.0mg L ^-1 +NAA 0.1mg L ^-1 ;

(3) Rooting culture step: cut the cluster buds and transfer ^them to the rooting medium for light culture to induce rooting to become test ^- tube plantlets. Based on MS basic medium, supplemented with calcium 440mg L ^-1 , sucrose 20g L ^-1 , pH value 7.0, and additionally added IBA 1.0mg L ^-1 . The test-tube seedlings were transplanted after being cultured on the medium for 1 month;

(4) Transplanting steps: transplant the test-tube seedlings with long roots into the substrate transplanted into the sand basin, and place them in a moist, shady environment to cultivate. The substrate in the sand basin is based on limestone: vermiculite: sandstone=1 : 2:1 ratio configuration;

(5) Field transplanting step: plant the test-tube seedlings after the strong seedlings in the above-mentioned sand basins in the limestone cave environment, and plant together with the associated species moss.

Embodiment Three

Using the leaves of Primulina tabacum Hance collected from the Lianzhou underground river scenic spot as explants, the tissue culture propagation and field cultivation of Primulina tabacum Hance are carried out, and the specific steps are as follows:

(1) Induction culture step: Inoculate the explants of the leaves of Primula japonica plants into the induction medium for culture, the induction medium is MS basic medium, supplemented with calcium 300mg L ^-1 , sucrose 30g L ^-1 , pH value 7.0 As a basis, add BAP 1.0mgL ^-1 +NAA0.5mg L ^-1 , culture conditions: 20～30℃, 10μmol m ^-2 s ^-1 low light irradiation every day for 14 hours, to induce primula officinalis leaves For the formation of adventitious buds, the explants were cultured for 60 days, and after the explants could be induced to form adventitious buds, the culture was continued for 30 days.

(2) Cluster bud propagation step: transfer the adventitious buds to the propagation medium for cultivation, under the conditions of 20-30°C, 30 μmol m ^-2 s ^-1 light per day for 14 hours, and subculture once a month. The propagation medium is based on MS basic medium, supplemented with calcium 440mg L ^-1 , sucrose 30g L ^-1 , pH 7.0, and BAP 1.0mg L ^-1 +NAA 0.1mg L ^-1 ;

(3) Rooting culture step: cut the cluster buds and transfer ^them to the rooting medium for light culture to induce rooting to become test ^- tube plantlets. Based on MS basic medium, supplemented with 440 mg L ^-1 calcium, 30 g L ^-1 sucrose, pH 7.0, and added 0.2% activated carbon. The test-tube seedlings were transplanted after being cultured on the medium for 1 month;

(4) Transplanting steps: transplant the test-tube seedlings with long roots into the substrate transplanted into the sand basin, and place them in a moist, shady environment to cultivate. The substrate in the sand basin is based on limestone: vermiculite: sandstone=1 : 3:1 ratio configuration;

(5) Field transplanting step: plant the test-tube seedlings after the strong seedlings in the above-mentioned sand basins in the limestone cave environment, and plant together with the associated species moss.

Claims (5)

1. a kind of Primulina tabacum Hance (Primulina tabacum Hance) tissue culture propagation and field cultivation method, it is characterized in that with the Primulina tabacum Hance plant blade of collecting in the field as explant, carry out cultivation propagation and cultivation according to the following steps: (1) Induction culture step: the leaf explants of Primula moss plant are inserted into the induction medium for culture, and the somatic embryogenesis or/and adventitious bud formation of the Primula moss leaf explants are induced. Cultivate in the dark at ℃ or irradiate with low light at 1-20 μmolm ^-2 s ^-1 for 12-14 hours per day, and cultivate for 50-70 days. After the explants can induce the formation of somatic embryos or adventitious buds, continue to cultivate for 20-30 day, the induction medium is based on MS basic medium, supplemented with calcium 220-440 mg L ^-1 , sucrose 20-30 g L ^-1 , pH 6.5-7.5, and NAA 0.5-1.0 mg L ^-1 +TDZ 0.5～1.0mgL ^-1 , or BAP 0.5～1.0mgL ^-1 +TDZ 0.2～1.0mgL ^-1 , or BAP 0.5～1.0mgL ^-1 +NAA 0.5～1.0mgL ^-1 ; (2) Cluster bud propagation step: transfer somatic embryos or adventitious buds to the propagation medium for culture, the conditions are 20-30°C, 20-50 μmol m ^-2 s ^-1 light per day for 14 hours, one month to Subculture once every one and a half months. The breeding medium is based on MS basic medium, supplemented with calcium 220-440mg L ^-1 , sucrose 20-30g L ^-1 , pH value 6.5-7.5, and BAP 0.5- 1.0mg L ^-1 +NAA 0.1～0.5mg L ^-1 ; (3) Rooting stage: Cut the cluster buds and transfer them to the rooting medium for light culture to induce rooting to become ^{test -} tube plantlets. The base is based on MS basic medium, supplemented with calcium 220-440mg L ^-1 , sucrose 20g L ^-1 , pH value 6.5-7.5, IBA 0.2-1.0mg L ^-1 and/or 0.2% activated carbon, The test-tube seedlings were transplanted after being cultured on the medium for 1 month; (4) Transplanting stage: the test-tube seedling of long root is transplanted in the substrate that is transplanted into the sand basin, is placed in the cultivation under the moist, shady environment, and the substrate in the sand basin presses limestone: vermiculite: sandstone=( 1～2):(2～4):(1～2) ratio configuration; (5) Field transplanting stage: the test-tube seedlings after the seedlings have been strengthened in the above-mentioned sand basins are planted in the limestone cave environment, and planted together with the associated species moss. 2. Primulina tabacum Hance (Primulina tabacum Hance) tissue culture propagation and field cultivation method according to claim 1, is characterized in that described induction medium is with MS basic medium, additional calcium 440mg L ^-1 , sucrose 20 ～30g L ^-1 , based on pH value 6.5～7.0, plus NAA 0.5mg L ^-1 + TDZ 1.0mg L ^-1 , or BAP 0.5mg L ^-1 + TDZ 0.2mg L ^-1 , or BAP 1.0 mg L ^-1 +NAA 0.5 mg L ^-1 . 3. Primulina tabacum Hance (Primulina tabacum Hance) tissue culture propagation and field cultivation method according to claim 1, is characterized in that described propagation medium is with MS basal medium, additional calcium 440mg L ^-1 , sucrose 20 ～30g L ^-1 , based on pH value 6.5～7.0, additionally add BAP 1.0mg L ^-1 +NAA 0.1mg L ^-1 . 4. Primulina tabacum Hance (Primulina tabacum Hance) tissue culture propagation and field cultivation method according to claim 1, is characterized in that described rooting medium is with MS basic medium, additional calcium 440mg L ^-1 , sucrose 20 ～30g L ^-1 , based on pH value 6.5～7.0, additionally add IBA 1.0mg L ^-1 and/or 0.2% activated carbon. 5. Primulina tabacum Hance (Primulina tabacum Hance) tissue culture propagation and field cultivation method according to claim 1, is characterized in that in the transplanting stage, the substrate in the sand basin transplanted by test-tube seedling is by limestone: vermiculite : Sand and stone = 1:2:1 ratio configuration.