阅读说明：本技术 一种长山药复壮种苗制备方法 (Preparation method of rejuvenating seedlings of Dioscorea polystachya ) 是由 尚勇越 尚勇进 于 2021-01-21 设计创作，主要内容包括：一种山药复壮种苗制备方法,其目的是效果稳定、操作简单、容易掌握；本发明应用植物组织培养技术使脱除病毒的山药微茎尖生长成苗后,通过继代培养使基数增大并在试管内生产微型零余子,收获后的零余子经过物理和化学复合处理后破除休眠,栽种到试管外的基质中发芽并生长成苗；所述继代培养是指长山药试管苗在生长为至少三节以上时,将其剪切为单节的切段,插入无菌培养基中进行培养,这些单节的茎段由腋芽生长为植株的茎叶部分,茎段下部生根生长成试管苗,如此反复不断扩大试管苗的数量。(A preparation method of a Chinese yam rejuvenation seedling, which aims to achieve stable effect, simple operation and easy mastering; the method comprises the steps of growing virus-free Chinese yam micro stem tips into seedlings by using a plant tissue culture technology, increasing the base number by subculture, producing miniature bulbil in a test tube, breaking dormancy of the harvested bulbil after physical and chemical compound treatment, and planting the miniature bulbil in a matrix outside the test tube to germinate and grow into seedlings; the subculture is to cut the Chinese yam into single-section cut sections when the Chinese yam test-tube plantlets grow into at least three sections, insert the cut sections into a sterile culture medium for culture, grow the single-section stem sections into stem and leaf parts of plants from axillary buds, root the lower parts of the stem sections to grow into the test-tube plantlets, and repeatedly and continuously expand the number of the test-tube plantlets.)

1. A method for preparing rejuvenated yam seedlings is characterized in that after virus-free yam micro-stem tips grow into seedlings by applying a plant tissue culture technology, the base number is increased by subculture, miniature bulbil is produced in a test tube, the dormancy of the harvested bulbil is broken after the harvested bulbil is subjected to composite treatment by physical and chemical methods, and the harvested bulbil is planted in a matrix outside the test tube to germinate and grow into seedlings.

2. The method for preparing rejuvenated yam seedlings according to claim 1, wherein the plant tissue culture technique is to perform virus removal and propagation in vitro by conventional aseptic technique on the yam stock, and the basic method comprises sterilizing the surface of the stem segment of the cut or bulbil of the reed head and tuber of yam, inoculating the stem segment into an aseptic culture medium for primary culture and secondary propagation, transitionally transplanting the rooted test-tube seedlings into a vermiculite matrix in a greenhouse/net room, maintaining humidity and periodically applying nutrient solution, and transplanting the seedlings to the field for conventional management when the seedlings grow to more than 20 cm; or generating bulbil in the test tube, breaking dormancy of the harvested bulbil, and growing into seedlings.

3. The method for preparing rejuvenated yam seedlings according to claim 1 or 2, wherein the production of miniature bulbil in a test tube is that the stem of yam vine is sterilized by surface disinfection, cut into single buds and inserted into a primary culture medium: MS + BA (6-benzylaminopurine) 0.5 mg/L + NAA (alpha-naphthylacetic acid) 0.1mg/L + agar 7g/L, adjusting pH value to 5.8 with dilute hydrochloric acid or sodium hydroxide solution, cutting into single bud after the seedling grows to five leaves, inoculating into subculture medium: MS + KT (kinetin) 2 mg/L + NAA (alpha-naphthylacetic acid) 0.01mg/L + activated carbon 0.5 g/L + agar 7g/L, adjusting pH value to 5.8 with dilute hydrochloric acid or sodium hydroxide solution,

around 7 roots can be grown around the cultivation, the seedling height is about 7 cm, and 2-4 bulbils with the diameter larger than 5mm are produced; and after the bulbil is harvested, continuously carrying out subculture on the test-tube plantlet, and thus continuously producing the bulbil.

4. The method for preparing rejuvenated yam seedlings according to claim 1 or 2, wherein the dormancy breaking of the harvested bulbil after the physical and chemical compound treatment means that the miniature bulbil from the test-tube plantlet is dormant like yam tubers, and the harvested bulbil does not have dormancy or the dormancy stage is insufficient, but can not germinate although the miniature bulbil can root underground, so that a new yam plant is difficult to grow, the miniature bulbil needs to be physically or chemically treated to break the dormancy to promote germination, and specifically, the miniature bulbil harvested in the test-tube is soaked in the following solutions: 100 mg/L of GA3 (gibberellin), 10 mg/L of BA (6-benzylaminopurine) and 770.02 percent of silwell wet are placed into a filter for suction filtration for three minutes, then the filter is taken out and placed at room temperature for 2 hours, and finally the bulbil is naturally dried in the shade at room temperature and is placed into a glass bottle to be stored at the low temperature of 4 ℃ for 2 months.

5. The method for preparing rejuvenated yam seedlings according to claim 1 or 2, wherein said subculture is performed by cutting a test-tube plantlet of yam into single-node cut segments when the test-tube plantlet grows into at least three segments, inserting the cut segments into a sterile culture medium, culturing the single-node cut segments, wherein the stem segments of the single-node cut segments grow into stem and leaf parts of the plant from axillary buds, and the lower parts of the stem segments root and grow into test-tube plantlets, thereby repeatedly and continuously increasing the number of the test-tube plantlets.

6. The method for preparing Chinese yam rejuvenation seedlings according to claim 1 or 2, characterized in that the substrate outside the test tubes is the substrate used for excessively transplanting the Chinese yam test tube seedlings outside the test tubes, and the substrate used in the method is a mixture of turf, vermiculite and perlite, and the ratio of the turf, the vermiculite and the perlite is 3:1: 1.

Technical Field

The invention relates to the field of agricultural seedling breeding, in particular to a yam seedling preparation method.

Background

The medicinal and edible parts of the long Chinese yam are rhizomes, the underground growth of the long Chinese yam is vertical and downward to more than one meter, the traditional planting and harvesting method is labor-consuming and time-consuming, so that the long Chinese yam is mostly scattered and planted in small scale and becomes the bottleneck of large-scale planting, the problem is partially solved by the research and development and the use of the existing special machinery, and conditions are provided for large-scale planting. In this case, superior germplasm becomes a primary factor. The quality of seedlings seriously influences the yield, and the current yam seedling preparation methods mainly comprise three methods: firstly, using yam head, taking a section with buds of a tuber, wherein the section is 20-40 cm long and is called a reed head, secondly, using underground succulent tuber of yam to cut the tuber into sections according to 8-l 0cm, and generating stem from hidden buds on the tuber; thirdly, the Chinese yam bulbil is used, and the hidden bud on the Chinese yam bulbil can grow into vine stem. The three methods have advantages and disadvantages respectively, but the germplasm degradation cannot be completely avoided. The virus eliminating technology and tissue culture technology can restore germ plasm to rejuvenate, and the method includes eliminating virus from the germ plasm, growing in sterile culture medium to form test tube seedling, rooting, transplanting to greenhouse matrix, and maintaining proper humidity, temperature and light for over transplanting. However, because the test-tube seedlings are thin and weak, the high over-transplanting survival rate is difficult to maintain, the micro bulbil is generated by using the test-tube seedlings with virus removal, the dormancy of the harvested bulbil is broken, and the obtained bulbil is planted in a substrate or a field to germinate and grow, so that the method becomes a novel rejuvenation seedling preparation method.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the method for preparing the rejuvenation seedlings of Chinese yam, which has the advantages of stable effect, simple operation, easy mastering, high survival rate and low production cost.

The method of the invention adopts plant tissue culture technology to ensure that the virus-removed Chinese yam micro-stem tips grow into seedlings, the base number is increased through subculture and micro bulbil is produced in a test tube, the dormancy of the harvested bulbil is broken after the compound treatment of physical and chemical methods, and the harvested bulbil is planted in a matrix outside the test tube to germinate and grow into seedlings. The method specifically comprises the steps of growing virus-removed Chinese yam micro stem tips into sterile test-tube seedlings with the length of more than seven centimeters, cutting the Chinese yam micro stem tips into single-bud stem segments on a superclean workbench, re-inoculating the single-bud stem segments into a culture medium, and increasing the base number through multiple subculture propagation of the single-bud stem segments, wherein the base number is determined according to the space of a culture room and the production scale. In the process, the micro bulbil is continuously harvested, dormancy is broken after the micro bulbil is treated by a chemical and physical compound method, the micro bulbil is planted in a matrix such as vermiculite outside a test tube in batches, the matrix is kept at a proper temperature to enable the micro bulbil to germinate and grow, the proper humidity temperature and illumination are kept in the process, and the micro bulbil is moved to a field when the seedlings grow to about twenty centimeters.

The plant tissue culture technology is to detoxify and propagate Chinese yam protospecies in vitro by conventional aseptic operation, and the basic method is to perform surface sterilization on the reed heads and tubers of Chinese yam or the stem segments of bulbil, then inoculate the Chinese yam protospecies in an aseptic culture medium for primary culture and secondary culture, transfer rooted test-tube seedlings into a vermiculite matrix in a greenhouse/net room, maintain humidity and apply nutrient solution regularly, and transplant the Chinese yam protospecies to a field for conventional management when the seedlings grow to more than 20 centimeters.

The production of the miniature bulbil in the test tube refers to a plant nutrition propagule with the diameter of more than 5mm generated by the test tube seedling of the Chinese yam in a tissue culture bottle. The method comprises the following steps of cutting Chinese yam stem sections into single buds after surface disinfection and sterilization and inserting the single buds into a primary culture medium: MS + BA (6-benzylaminopurine) 0.5 mg/L + NAA (a-naphthalene acetic acid) 0.1mg/L + agar 7g/L, and adjusting pH to 5.8 with dilute hydrochloric acid or sodium hydroxide solution. Cutting the seedlings into single buds after the seedlings grow to five leaves, and inoculating the single buds into a subculture medium: MS + KT (kinetin) 2 mg/L + NAA (alpha-naphthylacetic acid) 0.01mg/L + activated carbon 0.5 g/L + agar 7g/L, and the pH value is adjusted to 5.8 by dilute hydrochloric acid or sodium hydroxide solution. The culture can take root 7 pieces around, the seedling height is 7 cm, and 2-4 bulbil with diameter larger than 5mm can be produced. After the bulbil is harvested, the test-tube plantlet is subcultured, and the bulbil is repeatedly produced in the way.

The dormancy breaking of the harvested bulbil after treatment refers to that the miniature bulbil from the test-tube plantlet promotes the germination of the hidden bud on the bulbil through a physical, chemical or biological method. The method comprises the following steps of soaking the micro bulbil harvested in a test tube in the following solutions: 100 mg/L of GA3 (gibberellin) + 10 mg/L of BA (6-benzylaminopurine) + sillwet 770.02%, placing a beaker or an open container containing the bulbil and the solution in a filter flask for vacuum filtration for three minutes, then placing the beaker or the open container in a natural state at room temperature for 2 hours, finally naturally drying the bulbil at room temperature in the shade, and placing the beaker or the open container in a glass bottle for storage at the low temperature of 4 ℃ for 2 months.

The subculture is to cut the Chinese yam into single-section cut sections when the Chinese yam test-tube plantlets grow into at least three sections, insert the cut sections into a sterile culture medium for culture, grow the single-section stem sections into stem and leaf parts of plants from axillary buds, root the lower parts of the stem sections to grow into the test-tube plantlets, and repeatedly and continuously expand the number of the test-tube plantlets.

The transition transplanting is a process of transplanting the sterile seedlings out of a culture bottle or in an extra-matrix of a test tube, and in the process, the seedlings gradually adapt to the external environment and are weak and strong, and proper temperature, humidity and illumination are needed.

The substrate outside the test tube is used for excessively transplanting the Chinese yam test tube seedlings outside the test tube, and the substrate used in the method is a mixture of turf, vermiculite and perlite in a ratio of 3:1: 1.

The method has the advantages of stable effect, simple operation, easy mastering and the like, and does not influence the propagation of the test-tube plantlet after the bulbil is collected. The solution treated zeros are stored at 4 degrees for 2 months to break dormancy, or stored for a longer time at this temperature to accumulate a certain number of zeros for batch planting. The mode avoids the step of excessively transplanting the young and tender test-tube seedlings, improves the survival rate and reduces the production cost for preparing the rejuvenated Chinese yam seedlings.

Detailed Description

Example 1:

taking a yam variety 'Taigu long yam' (the length of underground stem is about 120 cm) as a test material, planting bulbil harvested in a field in a turf matrix in a park of Shanxi Juxin Weiyu agricultural science and technology development Limited company in 2018, growing vines, taking stem segments, performing surface disinfection and sterilization by a conventional method, performing aseptic operation to cut the stem segments into single buds, and inserting the single buds into a primary culture medium: MS + BA (6-benzylaminopurine) 0.5 mg/L + NAA (a-naphthalene acetic acid) 0.1mg/L + agar 7g/L, and adjusting pH to 5.8 with dilute hydrochloric acid or sodium hydroxide solution. Cutting the seedlings into single buds after the seedlings grow to five leaves, and inoculating the single buds into a subculture medium: MS + KT (kinetin) 2 mg/L + NAA (alpha-naphthylacetic acid) 0.01mg/L + activated carbon 0.5 g/L + agar 7g/L, and the pH value is adjusted to 5.8 by dilute hydrochloric acid or sodium hydroxide solution. After 28 days of culture, 7 roots can be grown, the seedling height is 7 cm, and 2-4 bulbils with the diameter larger than 5mm are produced. After the bulbil is harvested, the test-tube plantlet is subjected to subculture, and thus the bulbil is continuously produced. The harvested Bulbilus Dioscoreae was soaked in the following solutions: GA3 (gibberellin) 100 mg/L + BA (6-benzylaminopurine) 10 mg/L + sillwet 770.02%, suction-filtered for three minutes, left to stand at room temperature for 2 hours in a natural state, then naturally dried in the shade at room temperature, and stored in a glass bottle at a low temperature of 4 ℃ for 2 months. The treated bulbil is planted in a plug tray filled with turf matrix in 1 month in 2019, and is placed in a greenhouse at about 25 ℃, the germination rate reaches 80% in one month, and the growth is good.

Example 2:

taking a variety of the short and coarse Chinese yam Taigu short and coarse Chinese yam I suitable for mechanical harvesting (the length of underground stem block is about 60 cm) as a test material, planting the bulbil harvested in a field in a grass carbon matrix in a park of Shanxi Juxin Weiyu agricultural science and technology development Limited company in 2019 for 1 month, taking stem segments after generating creeping stems, performing surface disinfection and sterilization by a conventional method, and cutting the stem segments into single buds by aseptic operation to insert the single buds into a primary culture medium: MS + BA (6-benzylaminopurine) 0.5 mg/L + NAA (a-naphthalene acetic acid) 0.1mg/L + agar 7g/L, and adjusting pH to 5.8 with dilute hydrochloric acid or sodium hydroxide solution. Cutting the seedlings into single buds after the seedlings grow to five leaves, and inoculating the single buds into a subculture medium: MS + KT (kinetin) 2 mg/L + NAA (alpha-naphthylacetic acid) 0.01mg/L + activated carbon 0.5 g/L + agar 7g/L, and the pH value is adjusted to 5.8 by dilute hydrochloric acid or sodium hydroxide solution. After 28 days of culture, 7 roots can be grown, the seedling height is 7 cm, and 2-4 bulbils with the diameter larger than 5mm are produced. The harvested Bulbilus Dioscoreae was soaked in the following solutions: GA3 (gibberellin) 100 mg/L + BA (6-benzylaminopurine) 10 mg/L + sillwet 770.02%, suction-filtered for three minutes, left to stand at room temperature for 2 hours in a natural state, then naturally dried in the shade at room temperature, and stored in a glass bottle at a low temperature of 4 ℃ for 2 months. The treated bulbil is planted in a plug tray filled with turf matrix in 1 month 2020, and is placed in a greenhouse at about 25 ℃, the germination rate reaches 85% in one month, and the growth is good.

Because the varieties of the Chinese yam are various, a plurality of varieties of the short and thick tubers exist, and the method does not test the varieties of the short and thick tubers. The method is used for testing two long Chinese yams, wherein a variety with a tuber length of 120 cm is used in example 1, a variety with a tuber length of 60 cm is used in example 2, although the difference of 60 cm, the method is effective in subculture proliferation, micro bulbil formation and bulbil dormancy breaking of the two varieties in a test tube, and the effects are not obviously different.