Application of tissue culture in crop improvement

Application of tissue culture in crop improvement

Presented by,M. Uma Maheshwari,

M. Phil., (Botany)

Introduction

Plant tissue culture comprises a set of invitro techniques, methods, strategies that are part of plant biotechnology. Tissue culture had been exploited to create genetic variability, to improve the health of planted material, increase the number of desired germplasms. Tissue culture are combined with molecular techniques, successfully used to incorporate specific traits through gene transfer. The culture of protoplasts, anthers, microspores, ovules and embryos used to create new genetic variation. Cell culture produced somaclonal and gametoclonal variants with crop improvement potential. The culture of single cells and meristems can be effectively used to eradicate pathogens thereby increasing the yield of the crops.

Application of tissue culture in crop improvement

Newer molecular and cellular technologies have a broad significant impact on crop improvement

The various application of tissue culture approaches to crop improvement are-

Breeding and biotechnologyWide hybridizationHaploidsSomaclonal variationMicro propagationSynthetic seedsPathogen eradicationGermplasm preservation

Breeding and biotechnologyPlant breeding separated in to two activities

Manipulating gene Plant evaluation

Controlled pollination of plants lead to specific crosses result in new generation performed better in the yield

Tissue–culture techniques is having significant impact in manipulating genetic diversity

More than 50 different species have already been genetically modified either by vector dependent (Agrobacterium) or vector independent (Biolistic, micro-injection and liposome) methods

Tissue culture techniques have been played a major role in the development of plant genetic engineering, especially in efficient gene transfer and transgenic plant recovery

Wide hybridizationA critical requirement for crop improvement is the

introduction of new genetic material in to the cultivated lines of interest, whether via single genes, through genetic engineering or multiple genes through conventional hybridization or tissue culture techniques

The process of fertilization can be blocked at any of the stages, resulting in the functional barrier to the hybridization and the blockage of gene transfer between the two plants.

Two types of barriersPre-zygotic barriersPost-zygotic barriers

To overcome these problems, the following procedures arefollowed

in vitro fertilizationEmbryo cultureProtoplast fusion

in vitro fertilization

IVF has been used to facilitate both interspecific and intergeneric crosses, to overcome physiological based self compatibility and to produce hybrids

A wide range of plants have been recovered such as

Tobacco Clover Corn Rice Canola Cotton Poppy

Embryo culture It deals with the sterile isolation and in vitro growth of a mature or

an immature embryo of obtaining a viable plantSeed dormancy can be successfully bye passedTwo types of embryo culture

Mature embryo culture Immature embryo culture/ Embryo rescue

Mature embryo cultureo Mature embryos are isolated from ripe seeds and

are cultured in vitroo It is carried out during following condition

o Embryos remain dormant for long timeo Low survival of embryos in vivoo To avoid inhibition in seed germinationo Convert sterile seeds in to viable seedlings

Embryo culture

It has been successfully applied to a number of agricultural crops including cotton, barley, banana, orchids, roses, tomato, rice, jute and some brassicas

Application of embryo cultureo Prevention of embryo abortiono Overcoming seed dormancyo Shortening of breeding cycleo Production of haploidso Overcoming seed sterilityo Clonal propagation

Embryo rescueo It involves the culture of immature embryos

to rescue them from unripe or hybrid seeds that fails to germinate

Protoplast fusionIt has been suggested as a means of developing

unique hybrid plants which cannot be produced by conventional hybridization

Protoplasts of two plants can be fused by chemical or physical methods

Application of protoplast fusionIt has opened new possibilities for the in vitro genetic manipulation of to improve the crops Disease resistance Environmental tolerance Quality charactersCytoplasmic male sterility

Protoplast fusion

Haploids Haploid plants are characterized by possessing only a

single set of chromosomes. Haploid plants are of great significance for the production

of homozygous lines

At least 171 plant species have been used to produce haploid plants by pollen, anther and microspore culture Cereals (barley, maize, rice, rye, wheat) Fruits (grape and strawberry) Oil-seeds (rape and canola) Trees (apple, litchi, poplar and rubber) Plantation crops(cotton, sugarcane and tobacco) Vegetable crops (asparagus, Brussels sprouts,

cabbage, carrot, pepper, potato, sugar beet, tomato and wing bean)

Haploids

Androgenesis Haploid production

occurs through anther or pollen culture

In the plant biotechnology programme, haploid production is achieved by two methods

Androgenesis Gynogenesis

Gynogenesis Haploid production

occurs through ovary or ovule culture

Pollen culture preferable than anther culture because• Androgenesis, starting from a single cell can be

regulated• Isolated micro spores are ideal for several genetic

manipulations (transformation, mutagenesis)• The yield of haploid plants is relatively higher• Undesirable effect of anther wall and associated

tissues can be avoided

Somaclonal variation The genetic variations found in the in vitro cultured cells

are collectively called somaclonal variations. The plants are called somaclones

It occurs due to genetic heterogeneity in plant tissue cultures Expression of genetic disorders Spontaneous mutation due to culture conditions

The genetic changes include

Polyploidy Aneuploidy Chromosomal

breakage Deletion Translocations Gene

amplification Several

mutations

Many of the changes observed in plants are regenerated in vitro have potential agricultural and horticultural significance

The alterations include Plant pigmentation Seed yield Plant vigour and Size Essential oils Leaf and flower morphology Fruit solids Disease tolerance or

resistance

Such variations are observed in wheat, maize, tomato, sugarcane, oats, oil seed rape, celery, potato

Somaclonal variation

Micro propagationin vitro clonal propagation through

tissue culture is called micro propagation

The micro propagation technique is preferred to conventional asexual propagation

Small amount of tissue is neededPossible alternative for developing

resistanceMeans for international exchange

of plant materialsin vitro stock can be easily

proliferatedMicro propagation involves in vitro clonal propagation by two approaches

Multiplication by axillary buds/apical budsMultiplication by adventitious roots

Stage 0 • Selection of mother plant and its maintenance

Stage 1 • Initiation and establishment of culture

Stage 2

• Multiplication of shoots or rapid somatic embryo formation

Stage 3 • in vitro germination of somatic embryos and or rooting of shoots

Stage 4• Transfer of plantlets to

sterilized soil for hardening under green house condition

Major stages involved in Micro propagation

Micro propagation Applications

High rate of plant propagation

Production of disease-free plants

Production of seeds in some crops

Cost-effective process Automated micro

propagation

Disadvantages x Contamination of culturesx Brewing of mediumx Genetic variabilityx Vitrification x Cost factor

Synthetic seeds

Usually, Sodium or Calcium alginate is selected for encapsulation because it is less toxic to embryos and easy to handle

The artificial seeds can be maintained in a viable state still they are planted

A synthetic or artificial seed has been defined as a somatic embryo encapsulated inside a coating and is considered to be analogous to a zygotic seed

Different types of synthetic seeds areSomatic embryos encapsulated in a water gelDried and coated somatic embryosDried and uncoated somatic embryosSomatic embryo suspended in a fluid carrierShoot buds encapsulated in a water gel

Pathogen eradicationPlant species are infected with pathogens-viruses, bacteria, fungi,

mycoplasma and nematodes that causes diseasesTissue culture techniques employing meristem-tips are

successfully used for the production of disease-free plants

List of plants with virus-elimination by meristem-culture

Plant species Virus eliminatedSolanum tuberosum

Leaf roll potato virus-A, X, Y, S

Allium sativum Mosaic virusPetunia spp. Tobacco mosaic virusMusa spp. Cucumber mosaic virusBrassica oleracea

Cauliflower / turnip mosaic virus

Armoracia rusticena

Turnip mosaic virus

Germplasm preservation

Germplasm broadly refers to the hereditary material transmitted to the offspring through germ cells

The main objective of germplasm conservation is to preserve the genetic diversity of a particular plant or genetic stock for its use at any time in future

in vitro methods employing shoots, meristems and embryos are ideally suited for germplasm conservationThree main approaches

Cryopreservation Cold storage Low-pressure and Low-

oxygen storage

Advantages Large quantities can be stored in

a small areaMaintained in an environment

free from pathogensProtected against nature’s

hazardsFrom germplasm stock, large

number of plants can be obtained whenever it is needed

Obstacles for their transport through national and international borders are minimal (since they are maintained under aseptic condition)