“Minds Together” Sysmex Knowledge Congregation – Ploidy Webinar
In our endeavour to share knowledge, Sysmex India brings to you a series of webinars related to Industrial application.
Flow cytometry is the state of the art method in the agriculture industry for determination of ploidy level, genome size and pollen viability in plants.
DETECTION OF ANEUPLOIDS, HAPLOIDS AND TETRAPLOIDS IN BANANA AND OIL PALM
Dr Ramajayam D
Principal Scientist, ICAR – National Research Center for Banana
FLOW MINING IN KARANJ AND OTHER BIOFUEL CROPS
Dr Latha Rangan
Professor, Dept of Bioscience and Bioengineering, IIT Guwahati
PLOIDY BREEDING AND ROLE OF FLOW CYTOMETRY
Dr Arun Agarwal
Head, Biotechnology, Acsen HyVeg Pvt Ltd.
Banana is a polyploidy crop with 2n, 3n and 4n ploidies, owing to gamete restitution. Therefore, mixoploidy and unexpected polyploidies are very common in the banana anther culture as well as in the conventional banana breeding. Furthermore, formation of chimeras are very common during in vitro chromosome doubling of banana shoot cultures. During the study, more than 500 individual plants generated through (i) anther culture, (ii) induced tetraploids from embryogenic cell suspension (ECS) culture using oryzalin, and (iii) conventionally bred inter-specific banana hybrids were screened by flow cytometry (Cube 8, Sysmex). We have successfully identified tetraploids from anther culture (33 Nos.), ECS (8 Nos.) and conventional breeding (22 Nos.). Additionally, we have also identified diploids, triploids and mixoploids (>15 Nos.) from the said population. The number of plant material analysed by flow cytometry is much higher than by chromosome counts. Though we analysed all the plants using stomata measurements, it gave inconsistent results owing to different genomic constitution (i.e A & B genome). Thanks to flow cytometry, cytochimeras directly identified at in vitro level were discarded before weaning in glasshouse. Furthermore, flow cytometry allowed a better selection since some clones evaluated as tetraploid by chromosome counts were revealed to be aneuploids (chimeras) after one year in the field. Therefore, flow cytometry is less time consuming than classical cytology/stomata measurements and fits well for early mass screening of the polyploid bananas.
Haploids (Hs) are extremely valuable in plant breeding when they are converted to fertile doubled haploids (DHs) as they are 100% homozygous and may be used as parental lines to produce uniform F1 hybrids in oil palm. Frequency of spontaneous haploids in nature is too low in oilpalm, which has necessitated the development of an effective high-throughput screening system for detection of haploids. We used a more direct approach, i.e. a high-throughput flow cytometry (Partec Flowcytometer) method that detects naturally occurring Hs/noneuploids in oilpalm by measurement of DNA content of the cell nuclei when released into an analyte containing a DNA-specific dye. Spontaneously formed haploids have been detected from exotic germplasm collection, indigenous germplasm collection and from a primary nursery, raised from abnormal sprouts. The results suggest that the flow cytometry can be used ‘aggressively’ in screening ploidy level of off-types and for classification of oilpalm cultivars (tenera, dura and pisifera) at the nursery stage, and ‘defensively’ to assess and control seedling quality.
Many plants have been identified as potential biofuel crops to meet the increased demand for energy. As researchers attempt to exploit and improve the traits of significance, knowledge of genome size and DNA content becomes critical. Estimation of nuclear DNA content could be helpful in understanding genome organization and evolution. In this study, flow cytometric investigation has been done to estimate the genome size of four socio-economic non-edible oil crops viz. Pongamia pinnata, Jatropha curcas, Ricinus communis, and Mesua ferrea. The genome size / 2C nuclear DNA content were in the following order: J. curcas (0.86 pg/2C DNA) < R. communis (1.01 pg/2C DNA) < M. ferrea (1.52 pg/2C DNA) < P. pinnata (2.49 pg/2C DNA). Also, an attempt was made to see if any correlation existed between that of the nuclear DNA content and temperature in twelve individuals of the four biofuel crops. This knowledge will facilitate the use of available germplasm resources, enabling the development of optimized breeding strategies towards improvement in yield and production particularly as it relates to the oil trait.
Polyploids are organisms with multiple sets of chromosomes in excess of the diploid number. Polyploidy is common in nature and provides a major mechanism for adaptation and speciation. Approximately 50-70% of angiosperms, which include many crop plants, have undergone polyploidy during their evolutionary process. Polyploids could be game changer in future. Cotton is one of the best example presently. Flowering plants form polyploids at a significantly high frequency of 1 in every 100,000 plants, which leads to development of large sized, intense color, with high fragrance flowers. Over the last several decades, breeders have increased the world food production by utilizing the concept of heterosis in hybrid cultivars and use of polyploids. High frequencies of chromosome mutations are desirable in modern breeding techniques. One of the immediate and obvious consequences of polyploidy in plants is an increase in cell size which in turn leads to enlarged plant organs like potato, wheat, pear, water melon etc. Polyploidy has also been applied in breeding to develop disease resistant plants through the addition of extra chromosomes into the progeny genome. Chromosome doubling is reported to have an apparent effect on many physiological properties of a plant. The most discernable of these has been the increase in secondary as well as primary metabolism. Without flow-cytometry, it is very difficult to confirm polyploids at commercial level.
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