Let us make in-depth study of the meaning, principle, protocol, importance and applications of the root culture.
What is the Meaning of Root Culture?
Root culture can be defined as the culture of excised radical tips of aseptically germinated seeds in a liquid medium where they are induced to grow independently under controlled conditions.
Intact in vivo plants are not suitable for the isolation of intact root tips because the roots of ‘the plant are buried deeply in the soil. Again, root tips from young seedlings are very sensitive to toxic sterilants. So it is better to avoid the surface sterilization of young root tips for the establishment of root cultures. Root cultures can be successfully initiated from the excised radicle tips of aseptically germinated seeds.
Root tip cultures are generally maintained in moving liquid medium. In culture, root tips are induced to grow like that of root system of an intact plant. A clone of excised roots can also be established from a single root culture by repeatedly cutting and transferring of the main root tips or of lateral tips into fresh medium in every subculture at the interval of definite period. Growth of excised roots can be expressed in terms of fresh and dry weight, increase in length of the main axis, number of emergent laterals and total length of laterals per culture.
Initiation of Isolated Root Culture:
(1) Seeds are surfaced sterilized by the conventional methods and germinated on moist filter paper or White’s basal medium at 25°C in the dark (Fig 2.1).
(2) When the seedling roots are 20 to 40 mm in length, 10 mm apical tips (tip inoculum) are excised with a scalpel and each transferred to 40 ml of liquid medium contained in 100 ml wide-necked Erlenmeyer flasks.
(3) Flasks are incubated at 25°C in the dark.
Initiation of Clones:
The root material derived from a single radicle tip could be multiplied and maintained in continuous culture. Such genetically uniform root cultures are referred to as a clone of isolated roots. Initiation of root clones is a very simple technique.
The protocol is given below:
(1) Establish a root culture from a radical tip of a seed as described above.
(2) Transfer a 10-day-old established root culture to a sterile petri dish containing sterile medium. Next, using flamed scissors, cut the main axis of root into a number of pieces (each piece is called sector inoculum or initial), each bearing four or five young laterals.
(3) Transfer the individual sector inoculum aseptically to a flask liquid medium and incubate in dark at 25°C.
(4) Such sector culture can be used to initiate further tip culture using 10mn apical tips of laterals of a growing sector inoculums or the growing sector is again cut into 4-5 sectors to initiate the sector culture.
Importance of Root Culture:
The root of many species cannot be cultured. Studies with successful species (Tomato, Pea, Clover, Carrot, etc.) have contributed a lot of significant information’s.
The importance of root culture is given below:
Importance of Root Culture in Relation to Basic Information:
(1) Root cultures have increased our knowledge of carbohydrate metabolism, role of mineral ions, vitamins etc. in root growth.
(2) Root cultures have provided basic information regarding the dependence of roots on shoots for growth hormones.
(3) Root clones are ideally suited for the study of the effect of various compounds on root growth.
Specific Applications of Root Culture:
Study of Nodulation of Leguminous Root in Culture:
The process of nodule formation on the roots of leguminous plant by the nitrogen-fixing (NIF) bacteria (Rhizobium sp.) is a complex physiological system which is poorly understood. Root cultures of leguminous plant provide an ideal system to study it. When bacteria are inoculated directly in root culture, they quickly grow and spoil the whole culture.
Again, nitrate in the medium is required for the root growth but is inhibitory to nodulation. To overcome such difficulties M Raggio, N Raggio and J G Torrey modified the root culture technique for nodulation study. In their study, the base of an excised root of Phaseolus vulgaris was supplied with sucrose and vitamins via agar medium in a glass vial.
The remainder of the root was in contact with an inorganic nitrate-free medium containing Rhizobium. By this process, isolated roots develop nodules in culture. Therefore, in vitro nodulation helps to understand the relationship between symbiotic NIF bacteria and higher plants.
Regeneration of Shoots on Roots:
Culture of isolated roots can be maintained continuously for many years. However, in some species e.g. Atropa, Convolvulus arvensis, shoots can be induced to regenerate from cultured roots. The shoot primordia can be derived from callus at the cut ends of the roots, as in case of Atropa or endogenously from the internal tissues of the root as in Convolvulus. This phenomenon is of practical value as well as theoretical interest.
Study of Synthesis of Secondary Metabolites from Root Culture:
The roots of many medicinally important plant species synthesize pharmaceutical important alkaloids as by-products of normal metabolism (secondary metabolites). Root culture have been used to locate the site of biosynthesis of such compounds. Root culture techniques are also used to increase the synthesis of such compounds in cultured root by some nutritional manipulations.
Initiation and Development of Secondary Vascular Tissues:
Normally excised cultured roots show only the primary structure of young seedling radicle and, therefore, do not form secondary vascular tissue. But it has been found that excised root tips from pea seedling develop a vascular cambium when cultured in medium containing indoleacetic acid. Attempts have also been made to define the factors that determine the site, time of origin and functioning of such vascular cambium.
Torrey studied this phenomenon using a modification of the technique developed by Raggio and Raggio. The basal 5 mm portions of excised 15 mm long pea root were inserted into an agar solidified basal medium supplemented with various test substances e.g., auxins, cytokinins, mesoinositol and extra sucrose and the exposed 10 mm portion of the root was placed in a petri dish of basal agar medium containing inorganic salts, vitamins and sucrose.
When the bases of pea roots were fed with extra sucrose (8%) and IAA (10-6M) a vascular cambium was initiated. But when the whole portion of roots was allowed to grow in a basal medium, they did not form secondary vascular tissues. These experiments have suggested that auxins, cytokinins, mesoinositol extra sucrose may have an important role in cambial development.