What is the aseptic cultivation method?

Aseptic cultivation is a method in which plant tissues are enclosed in an aseptic environment and cultivated with nutrient solutions. It usually uses plant callus or cut stems to replicate plant genes, and they can grow and become the new plant with the same gene. So it is also known as in vitro propagation. The characteristic of this kind of cultivation was it can choose the plant tissues that have excellent genes to cultivate the excellent plant. And because the roots of plants directly contact nutrients, that the growth rate is faster than soil cultivation! In botany, there are also cases of using aseptic cultivation to regenerate plants that are about to become extinct ^[3].

The operation method of aseptic cultivation

Step 1. Plant Tissue Culture

Use plant roots, stems, leaves, and others which has the ability to self-repairing healing tissues, inoculate them on plant culture media, and use cell culture to grow new buds ^[4]. According to the type of culture medium, it can be divided into the solid agar gel medium and liquid suspension medium.

Step 2. Rooting culture

After the new buds are released. It can be transplanted into the rooting culture medium to promote the healthy root development of the plant. It can also use the cut stem of the plant in the same method.

Step3. Domestication and cultivation^[5]

The healthy plants with intact roots are moved from the sterile culture container to the outside and planted in the Inert medium, such as rock wool, sponge, and gravel, etc., then continue to grow after being adapted to the atmospheric environment.

Further reading: Soilless culture system and precautions

Studies have shown that the choice of plant culture media has a great influence on the germination rate. During the cultivation process, the growth and metabolism of plant tissues and roots may also change the pH value of the medium. The pH value of the medium is very important for the growth of plants. Changes in pH value may slow down plant growth or even death. Therefore, we can adjust and maintain the pH of the culture medium by using biological buffers. The following is the list of common biological buffers used in plant tissue culture medium:

MES

Basic information:

• CAS No.: 4432-31-9
• Useful pH range: 5.5 – 6.7

Application of MES:

• According to the result of MES Buffer Affects Arabidopsis Root Apex Zonation and Root Growth by Suppressing Superoxide Generation in Root Apex research indicates the 0.01% and 0.1% of MES enhance root growth from the early developmental stage of seedlings, however, 1% MES inhibited.^[1]
• Some research has revealed that 1–2 mM of MES solution has an excellent capacity of buffering and it shows neither inhibition of nodulation nor lowering of nitrogen fixation in soybean hydroponic culture. ^[1]
• The research has shown that 5 mM of MES was no impact on the growth or uptake of most nutrients.^[1]
• The research has shown that Potassium uptake was even enhanced by the MES buffer in non-nodulated seedlings of soybean.^[1]
• In the research of the effects of pH, MES, arabinogalactan-proteins on microspore cultures in white cabbage, the combination of 10 mg l−1 gum arabic and 3 mM MES in NLN-13 at pH 6.4 significantly enhanced microspore embryogenesis efficiency.^[2]
• The provision of MES to plants provided with NH4 significantly increased plant yield and reduced nodule number at all oncentrations.^[9]
• In the research of the effect of pH on rooting of cut from stems of apple micro shoots. When using the MES as a buffering agent, the pH was stable when set at 5.0–6.5.^[10]
• The medium fortified with MES and proline had a significant impact on cell proliferation during the growth of rice suspension culture.^[11]
• It was observed that medium is supplemented with 500 mg l−1 of each PVP, Casein hydrolysate, and MES buffer showed high amounts of callus.^[11]
• In the research of the use of MES buffer in early nodulation studies with annual Medicago species, 0.5mM of MES solution can stable pH by daily titration and the use of a high nitrate to ammonium ratio (9: 1) in solution.^[12]
• It is used in Cannabis sativa photoautotrophic micropropagation, the results showed 300 mL per vessel of fertilizer solution containing 5-mM MES buffer stabilized medium pH and increased rooting success.^[13]

HEPES

Basic information:

• CAS No.: 4432-31-9
• Useful pH range: 6.8 – 8.2

Application of HEPES:

• In production of mycorrhizal inoculum by static culture hydroponics, HEPES is used in maintained the pH value of culture solution.^[8]

PIPES

Basic information:

• CAS No.: 5625-37-6
• Useful pH range: 6.1 – 7.5

Application of PIPES:

• In production of mycorrhizal inoculum by static culture hydroponics, PIPES is used in maintained the pH value of culture solution.^[8]

MOPS

Basic information:

• CAS No.: 1132-61-2
• Useful pH range: 6.5 – 7.9

Application of MOPS:

• In the optimization of hydroponic growth system research, MOPS buffer was used as a fixative solution of Pongamia pinnata roots. It has ability to soften tissue to cut the segments.^[7]
• In the optimization of hydroponic growth system research, MOPS buffer used as the Incubation buffer and the wash buffer of Pongamia pinnata roots.^[7]
• In NH4 nutrition studies it is desirable to use buffers with a pK, between 6.5 and 7.5 because of thelarge pH drop experienced. Because of the adverse effects of ADA and ACES, BES and MOPS are the most favoured alternatives.^[9]

MES buffer is widely used at Agricultural Biotechnology. It is important to notice that the choice of the buffer. when choosing phosphate buffer, plants will absorb phosphate. When choosing the buffer containing chlorine, high concentrations of chlorine may be toxic to plants.^[6]

Further reading: MES Buffer used in HEMP

Reference:

^[1] MES Buffer Affects Arabidopsis Root Apex Zonation and Root Growth by Suppressing Superoxide Generation in Root Apex https://www.frontiersin.org/articles/10.3389/fpls.2016.00079/full

^[2] Yuan, Sx., Su, Yb., Liu, Ym. et al. Effects of pH, MES, arabinogalactan-proteins on microspore cultures in white cabbage. Plant Cell Tiss Organ Cult 110, 69–76 (2012). https://link.springer.com/article/10.1007/s11240-012-0131-z

^[3] An effective nutrient medium for asymbiotic seed germination and large-scale in vitro regeneration of Dendrobium hookerianum, a threatened orchid of northeast India https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3260561/

^[4] Morphogenic processes in callus tissue cultures and de novo regeneration of plants in Actinidia chinensis Planch https://pbsociety.org.pl/journals/index.php/asbp/article/view/833

^[5] Purohit, S., Rawat, J.M., Pathak, V.K. et al. A hydroponic-based efficient hardening protocol for in vitro raised commercial kiwifruit (Actinidia deliciosa). In Vitro Cell.Dev.Biol.-Plant (2020). https://doi.org/10.1007/s11627-020-10127-3

^[6] How to maintain Hoagland Nutrient Solution(normally the pH is 5.6) at pH 3.0, 7.0 and 11.0 https://www.researchgate.net/post/How_to_maintain_Hoagland_Nutrient_Solutionnormally_the_pH_is_56_at_pH_30_70_and_110

^[7] Optimization of hydroponic growth system and Na+-fluorescence measurements for tree species Pongamia pinnata (L.) pierre https://doi.org/10.1016/j.mex.2020.100809

^[8] Production of mycorrhizal inoculum by static culture hydroponics https://patents.google.com/patent/WO1991001082A1/en

^[9] Nutrient Solution pH Control using Dipolar Buffers in Studies of Trifolium repens L. Nitrogen Nutrition https://www.researchgate.net/publication/240590018_Nutrient_Solution_pH_Control_using_Dipolar_Buffers_in_Studies_of_Trifolium_repens_L_Nitrogen_Nutrition

^[10] Effect of medium-pH and MES on adventitious root formation from stem disks of apple December 2008Plant Cell Tissue and Organ Culture 95(3):285-292 DOI: 10.1007/s11240-008-9442-5

^[11] Plant regeneration from cell suspension culture in Saccharum officinarum L. and ascertaining of genetic fidelity through RAPD and ISSR markers. https://link.springer.com/article/10.1007/s13205-016-0579-3

^[12] Ewing, M.A., Robson, A.D. The use of MES buffer in early nodulation studies with annual Medicago species. Plant Soil 131, 199–206 (1991). https://doi.org/10.1007/BF00009449

^[13] Cannabis sativa L. photoautotrophic micropropagation: a powerful tool for industrial scale in vitro propagation https://www.x-mol.com/paper/jourmal