Stock solution:-

Murashige and Skoog (IMS) medium was formulated in 1962 and the usage of
this media is considerably high compared to other types of plant media. This is because MS
medium was found to be the most suitable medium used for plant regeneration from
tissues and calluses. MS medium was invented by plant scientists Toshio Murashige and
Folke K. Skoog during Murashige's search for a new plant growth regulator. Initially, the
media was produced based on mineral analysis of tobacco. Since the MS media contains
high amount of nitrogen fixing agents, it was found out i that the MS medium was equally
compatible for other plant species as well.

To have a complete form of MS media, the major nutrients required were the
macronutrient, micronutrient, serum, vitamins and in most cases, presence of plant i growth
regulators or hormones gives off a better result of root and shoots formation. And in high
presence of these plant growth regulators, callus can be induced as well.

Preparation of stock solutions for MS medium is a vital step in media preparation. Several
nutrients must be prepared and stored in order to have a good working: medium.
Therefore, to have a working solution, the stock must be prepared beforehand.

MS) medium is the most suitable and commonly used basic tissue culture medium for plant
regeneration. Here we describe the components of the MS medium, stock preparation and
storage used In tissue culture. The commonly used plant growth regulator and antibiotics
used are also described. So it is convenient to prepare the concentrated stock solutions of
macro- salts, micro-salts, vitamins, amino acids, hormones etc. All stock solutions should be
stored in a refrigerator and should be checked visually for contamination with
microorganisms or precipitation of ingredients.

Stock solution of vitamins, amino acids and hormones should not be stored for indefinite
period and should be kept in a deep freezer chamber. The widely used culture medium was
formulated by Murashige and Skoog (commonly called MS medium), so the procedure for
the preparation of stock solution of MS medium (1962) is given below:

Particularly macro-salts should be made at ten/twenty times of (X10 or X20) their final con-
centration in the medium while micro-nutrients can be prepared at thousand time (X 1,000)
of their final strength. While making the stock solutions, it is advisable to dissolve each con-
stituent completely before adding another otherwise precipitation of salts may occur
Anther culture :-

Anther culture means plant regeneration from the haploid microspore cells with the aim of
haploid and dihaploid plant production. There are few reports on plant regeneration in the
anther culture of buckwheat. Adachi et al. (1988) the first regenerated whole plants in
anther culture of the diploid cultivar of common buckwheat Miyazaki-Zarai.
The regenerants were all diploid but their origin was unclear. The authors did not exclude
the chromosome duplication in plant regenerants. In the experiment of Kong et al.
(1992) regenerants were obtained in all six cultivars used. The adopted plants grew well but
did not set the seeds. Unfortunately, the number of chromosomes was not counted in this
case.
Bohanec et al. (1993) were the first who proved the presence of haploid plants among the
regenerants of diploid cv. Daria. They noted the strong tendency toward endoduplication
because they detected the presence of diploid, triploid, tetraploid, and aneuploid
cells. Berbec and Doroszewska (1998) induced regeneration in anther culture of one
tetraploid and two diploid cultivars.
The most prevalent class of regenerants was tetraploids. No haploids were found among
regenerants derived from the diploid cultivars; likewise no diploid regenerants were
obtained from the tetraploid cultivar. The authors assumed that polyploid regenerants must
have arisen through multiplication of the initial chromosome number in microspores or
somatic tissues. Yui and Yoshida (2001) obtained a number of plantlets regenerated in
anther culture of three diploid Japanese cultivars. Most of the plants showed the same
amount of DNA as that of the control and were considered to be diploid.
Wang and Campbell (2006) tried to apply the anther culture in the buckwheat breeding
process.
Among the seven genotypes tested, three were the self-pollinated lines developed
from interspecific hybridization between common buckwheat and wild F. homotropicum,
and two genotypes were F1 hybrids. Tetraploid and somatic diploid regenerants were
predominant and no haploid plants were found. Based on the somatic origin of the
regenerants the authors concluded that buckwheat is a difficult species in which to produce
haploids or doubled haploids via anther culture.
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.

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.

(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
Micro grafting :-
The transfer of small shoot apices onto rootstocks (termed micrografting), can
be carried out in vivo or in vitro. Navarro (1988) lists four uses which have been found for
the technique: • Obtaining plants free from specific virus and virus-like diseases;

• A method for separating virus and virus-like organisms in mixed infections;

• For studying graft incompatibility between scions and rootstocks, and the histological and
physiological aspects of grafting;

• A minimum risk method for importing plant material through quarantine.

Micrografting is thus indirectly useful in micropropagation: the necessary techniques are

usually too time consuming and the proportion of successful takes is generally too low, for it
to be of direct application.

The rootstocks used for micrografting are commonly newly-germinated seedlings, but it is
also possible to use rooted cuttings or micropropagated shoots. Before micrografting can be
carried out, it is necessary to prepare suitable rootstock material. When seedling rootstocks
are used, and all stages of grafting are conducted in vitro, seeds are surface sterilised and
germinated aseptically in vessels containing nutrient salts (e.g. those of MS medium). The
seedlings may be supported on agar medium or on a porous substrate, such as sterile
vermiculite, which allows the growth of a branched root system. Micrografting is then
effected by cutting off the tops of the seedling rootstocks and placing small shoot apices
onto the exposed surface. When grafts are successful, rootstock and scion grow together to
produce a plant. It is usually necessary to examine freshly grafted seedlings on a regular
basis and remove any adventitious shoot arising on or below the graft union.