Classification of Plant Tissue Culture Technique
I) Embryo Culture:
For embryo culture, embryos are excised from immature seeds, usually under a ‘hood’, which provides a clean aseptic and sterile area. Sometimes, the immature seeds are surface sterilized and soaked in water for few hours, before the embryos are excised. The excised embryos are directly transferred to a culture dish or culture tube containing synthetic nutrient medium.
Entire operation is carried out in the ‘laminar flow cabinet’ and the culture plates or culture tubes with excised embryos are transferred to a culture room maintained at a suitable temperature, photoperiod and humidity. The frequency of excised embryos that gives rise to seedlings generally varies greatly and medium may even have to be modified made for making Interspecific and Intergeneric crosses within the tribe Triticeae of the grass family. The hybrids raised through culture have been utilized for i) Phylogenetic studies and genome analysis. ii) Transfer of useful agronomic traits from wild genera to the cultivated crops and iii) to raise synthetic crops like triticale by producing amphiploids from the hybrids.
Embryo culture has also been used for haploid production through distant hybridization followed by elimination of chromosomes of one of the parent in the hybrid embryos cultured as above. A popular example includes hybridization of barley and wheat with Hordeum bulbosum leading to the production of haploid barley and haploid wheat respectively. Haploid wheat plants have also been successfully obtained through culture of hybrid embryos from wheat X maize crosses.
Application of Embryo Culture:
i) Recovery of distant hybrids.
ii) Recovery of haploid plants from Interspecific crosses.
iii) Propagation of orchids.
iv) Shortening the breeding cycle
v) Overcoming dormancy.
In addition ovule and ovary can also be cultured.
II) Meristem Culture:
In attempts to recovery pathogen free plants through tissue culture techniques, horticulturists and pathologists have designated the explants used for initiating cultures as ‘shoot –tip’, tip, meristem and meristem tip. The portion of the shoot lying distal to the youngest leaf primerdium and measuring up about 100 µm in diameter and 250 µm length is called the apical meristem. The apical meristem together with one to three young leaf primordia measuring 100-500 µm constitute the shoot apex. In most published works explants of larger size (100-1000 µm long) have been cultured to raise virus- free –plant. The explants of such a size should be infact referred to as shoot-tips. However, for purpose of virus or disease elimination the chances are better if cultures are initiated with shoot tip of smaller size comprising mostly meristematic cells. Therefore, the term ‘meristem’ or meristem-tip’ culture is preferred for in vitro culture of small shoot tips.
The in vitro techniques used for culturing meristem tips are essentially the same as those used for aseptic culture of plant tissues. Meristem tips can be isolated from apices of the stems, tuber sprouts , leaf axils , sprouted bunds o cuttings or germinated seeds.
Application of Meristem Culture:
i) Vegetative propagation
ii) Recovery of virus free stock.
iii) Germplasm exchange
iv) Germplasm conservation
III) Anther or Pollen Culture:
Angiosperms are diploid the only haploid stage in their life cycle being represented by pollen grains. From immature pollen grains we can sometimes raise cultures that are haploid. These haploid plants have single completes set of chromosomes. Their phenotype remains unmasked by gene dominance effects. In china, several improved varieties of plants have been grown from pollen cultures.
When pollen grains of angiosperm are cultured, they undergo repeated divisions. In Datura innoxia the pollen grains from cultured anther can form callus when grown on a media supplemented with yeast extract or casein hydrolysate. Similarly, when isolated anthers are grown on media containing coconut milk or kinetin, they form torpedo- shaped embryoids which in due course grow into small haploid plantlets. The usual approach in anther culture is that anthers of appropriate development stage are excised and cultured so that embryogenesis occurs. Alternatively pollen grains may be removed form the anther, and the isolated pollen is then cultured in liquid medium. Cultured anthers may take upto two months to develop into plantlets.
Pollen culture or anther culture is useful for production of haploid plants. Similarly, haploid plants are useful in plant breeding in variety of ways as follows:
i) Releasing new varieties through F1 double haploid system.
ii) Selection of mutants resistant to diseases.
iii) Developing asexual lines of trees or perennial species.
iv) Transfer of desired alien gene.
v) Establishment of haploid and diploid cell lines of pollen plant.
IV) Tissue and Cell Culture:
Single cells can be isolated either from cultured tissues or from intact plant organs, the former being more convenient than the latter. When isolated from culture tissues, the latter is obtained by culturing an organised tissue into callus. The callus may be separated from explant and transferred to fresh medium to get more tissue. Pieces of undifferentiated calli are transferred to liquid medium, which is continuously agitated to obtain a suspension culture. Agitation of pieces break them into smaller clumps and single cells, and also maintains uniform distribution of cells clumps in the medium. It also allows gases exchange.
Suspension cultures with single cells can also be obtained from impact plant organs either mechanically or enzymatically. Suspension cultures can be maintained in either of the following two forms i) Batch culture: are initiated as single cells in 100-250 ml flasks and are propagated by transferring regularly small aliquots of suspension to a fresh medium. ii) Continuous culture: are maintained in steady state for long periods by draining out the used medium and adding fresh medium, in this process either the cells separated from the drained medium are added back to suspension culture or addition of medium is accompanied by the harvest of an equal volume of suspension culture.
Application of Cell Culture:
i) Mutant selection
ii) Production of secondary metabolites or biochemical production.
iv) Clonal propagation
v) Somaclonal variations