Tomato Isolated Root Culture Technology

Root culture is mostly used to study root physiological metabolism, organ differentiation and morphogenesis. Root cell cultures can also be subjected to mutagenesis to screen out mutants for plant breeding. Therefore, the theoretical and practical research and development of isolated root culture is an important content of in vitro plant organ culture. The nutritional needs of isolated roots are generally consistent with the requirements of most plant tissue cultures, but there are also special features. For example, iodine and boron are important for the growth of isolated tomato roots, and the lack of these two trace elements will hinder the growth of isolated roots.

Principle

Under natural conditions, roots are mainly important organs for plant absorption and fixation. At the same time, the roots of some plants also have the function of reproduction. Plant roots grow fast, have strong metabolic capacity and small variation, which makes them of great theoretical and practical significance in the study of root nutrient absorption, changes in growth and metabolism, organ differentiation and morphogenesis.

According to the principle of plant cell totipotency, any cell (animal, plant, etc.) with a complete nucleus has all the genetic information (DNA) necessary to form a complete individual. As far as tomato root cells are concerned, they also have the ability to form complete regenerated plants. Such cultures of genetically consistent roots derived from a single root system and preserved by subculture are called asexual lines of isolated roots. The use of these asexual lines of isolated roots allows for experimental studies on organ regeneration systems, rapid propagation of seedling asexual lines and other aspects.

Figure 1. Steps of complete in vitro regeneration in S. lycopersicum cv. Riogrande. (Saeed, W.; et al. 2019)

Procedures

The isolated root grows under artificially controlled environment and nutritional conditions, and is affected by many factors such as genotype, culture conditions, hormones, etc. The specific steps of tomato isolated root culture are as follows:

A. Carry out the explant culture operation process on the sterile tomato root.

1. Put the tomato seeds into a 100 mL beaker and rinse 3 times with distilled water.
2. Under the sterile condition of the ultra-clean workbench, transfer the seeds to a sterile 100 mL beaker with sterilized tweezers, pour into70% alcohol, and sterilize the surface of the tomato seeds for 30 seconds.
3. Pour out the alcohol and pour into saturated bleach solution for 10 minutes, or sterilize in 0.1% to 0.2% mercuric chloride for 5 to 10 minutes, shake continuously during the disinfection process to fully sterilize it, and discard the disinfectant. Rinse 5 times with sterile water.
4. Transfer the seeds to a petri dish with sterile filter paper, remove excess water, and put the seeds into a triangular flask or a petri dish filled with MS basal medium under aseptic conditions.
5. The seeds are germinated under sterile conditions. After the roots elongate, take a root tip with a length of 1.0 cm from one end of the root tip and inoculate it in the medium. Cultures of these roots grow rapidly. Tomato roots grow about 1cm per day, and lateral roots develop after a few days. After 4 days of culture, lateral roots develop, and after 7 to 10 days, the root tips of lateral roots can be cut off as new culture materials for further expansion.
6. After the lateral roots grow for about 1 week, the root tips of the lateral roots can be cut out for expansion culture. They grow rapidly and grow lateral roots, which can be cut off and cultured again. By repeating this, asexual lines of isolated roots derived from a single root tip can be obtained.
7. The asexual lines of root segments were transferred to the differentiation medium for culture, and different concentrations of auxin and cytokinin were added to the medium.

B. Select normally growing plant roots, which must first be washed thoroughly with water due to their growth in the soil, and larger roots should be brushed with a soft-bristle brush. Try to select roots free from diseases and pests, cut off the damaged parts with a knife, and disinfect them after absorbing them with filter paper. First rinse with 70% alcohol, then soak in 0.1%～ 0.2% mercuric chloride for 5 to 10 min or placed in a 2% sodium hypochlorite solution for 10 to 15 min. Then rinse with sterile water 5 times, blot dry with sterile paper after rinsing, and cut the root tip for culture under sterile conditions.

Root tips obtained by either of the above methods are inoculated in the medium and lateral roots develop after a few days. After the lateral roots have grown for 1 week, the tips of the lateral roots can be taken for expansion and they will grow rapidly and produce lateral roots, which can be cut off again for culture. This can be repeated every 7 to 10 d to obtain asexual propagation lines with roots from a single root tip. Root segments can also be cut to proliferate and form primary and lateral roots to obtain asexual propagation lines of isolated roots.