Pollen Culture

Presented by Joshua Samuel I M.Sc Biotechnology DR.G.R.D.C.S.

Pollen from Cactus flower

Scanning Electron microscope of anther with pollen attached on them.

What is Anther or Pollen culture? Pollen culture (microspore culture) is a technique in which haploid plants are obtained from isolated pollen grains while in anther culture those are obtained from pollens, by placing anthers on a suitable, synthetic culture medium. The technique was discovered by Guha and Maheshwari (1964). It is one of the various tissue culture techniques or methods used. Progeny developed by this technique contains a single set of chromosomes.

Procedure for In-vitro Anther culture Collection of anthers Preferably should be taken from plants grown under controlled conditions or may be taken from plants grown in the field. Transfer of anthers to the culture medium Flower buds of the appropriate developmental stage are collected and surface sterilized. Anthers from the buds are excised and placed horizontally on surface (or partially embed) on culture medium. Important to note is, injury to anthers may induce callus formation from anther walls. To avoid this, small anthers are cultured as such and sometimes entire inflorescence is cultured.

Procedure for In-vitro Pollen culture Squeezing method Anthers (about 50 to 60) are placed in 20 ml of medium. Squeezed gently with a glass rod to separate pollens from anther walls. The prepared solution is filtered through a nylon mesh of suitable pore size. Filtrate is centrifuged for about 5 min at 500-800 rpm. The resulting pollen dense pellet is collected, washed twice. Final concentration of pollens is adjusted to 103-104 pollens/ml. Float culture Anthers are excised. Those are placed in petri dishes containing liquid medium. Anthers float in liquid medium. The anthers release their pollen grains into the medium in a few days.

Culture Medium for anther or pollen culture:

Medium used depends on various factors like Species Genotype of donor plants and anthers Age of donor plants and anthers Growing conditions of donor plants

Isolated pollen grains are cultured on a synthetic medium, the important ingredients of which are glutamine, L-serine and inositol. For anther cultures sucrose is essential ingredient.

Culture Environment Culture conditions are different according to certain conditions as we have listed under culture medium. Alternate light (12 to 18 hr; 5,000-10,000 lux) and dark periods (12-6 hr) at 22C.

Pathways of development Pathways of pollen development in anther culture or pollen culture are of the following. Pathway I Pathway II Pathway III Pathway IV

Pathway I Uninucleate pollen grain divides equally into two equal daughter cells. Both of these cells undergo divisions to give rise to a proembryonic mass. This proembryonic mass either directly (through embryo formation) or indirectly (through callus formation) may give rise to a haploid plant. Examples Datura

Pathway II Uninucleate pollen grain divides unequally to yield generative and vegetative cell. Callus / embryo originate from vegetative cell alone, while the generative cell degenerates immediately or after undergoing one or two divisions. Examples Datura, tobacco, wheat, barley, chillies. Pathway III

Uninucleate pollen grain divides unequally to yield generative and vegetative cell. Generative cell undergoes successive divisions to develop callus / embryo, while the vegetative cell does not divide or divides only to a limited extent forming a suspensor like structure
Examples Hyoscyamus niger.

Pathway IV Uninucleate pollen grain divides unequally to yield generative and vegetative cell. Callus / embryo develop by repetitive divisions of both of these cells. Examples Datura

Factors influencing pathways of development, in Anther or Pollen culture Genotype Growth hormones Culture medium Growth (physical) conditions Stage of pollen development Pretreatments

Applications of pollen or anther culture Haploid production Major application of pollen / anther culture is production of haploid plants. It takes much time to produce haploid plants by conventional breeding methods (many generations of inbreeding or backcrossing). As pollens are haploid, plants developed from these are homozygous (haploids). Protoplast isolation Used for protoplast isolation as single pollens (unicellular) are available. Transformation Used in transgenic plant formation, it can be done with less time consumption. Crop improvement In-vitro anther culture is used for improvement in vegetable and cereal crops e.g. asparagus, sweet pepper, watermelon, cabbage, broccoli, wheat etc.

Advantages of Anther or Pollen culture Simple Less time consuming, large number of haploids can be produces in less time. This topic is discussed in applications of anther and pollen culture. Responsive, most of the anthers used in culture respond. Pollen culture avoids the risk of chimera and callus formation from anther walls.

Disadvantages of Pollen or Anther culture Requires skill to remove anthers without causing damage, it is one of the most tedious jobs. Not much successful in case of cereal crops. Risk of chimera and callus formation from anther walls, in anther culture.

References 1. http://www.liv.ac.uk/~sd21/tisscult/case_study_1.htm 2. http://books.google.com/books/about/Plant_Tissue_Culture.html?hl=de &id=BEH1czIoVAgC 3. http://theagricos.com/tissue-culture/anther-or-pollen-culture/ 4. http://www.thefreedictionary.com/anther 5. http://www.biology-online.org/dictionary/Anther 6. http://en.wikipedia.org/wiki/Pollen 7. http://www.ifpindia.org/Identification-of-Pollen-Grains.html

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