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.
Application:
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.
iii) Biotransformation
iv) Clonal propagation
v) Somaclonal variations
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