RAPD, ISSR and RFLP fingerprints as useful markers to evaluate genetic integrity of micropropagated plants of three diploid and triploid elite tea clones representing Camellia sinensis (China type) and C. assamica ssp. assamica (Assam-India type)

Abstract

An efficient in vitro propagation method using enhanced axillary branching cultures produced plants from nodal explants of three mature, elite tea clones: diploid UPASI 26 and UPASI 27 (2n=2x=30) representing Camellia sinensis (China type) and triploid UPASI 3 (2n=3x=45) representing C. assamica ssp. assamica (Assam-India type). The genetic fidelity of the micropropagated plants of these three tea clones was assessed by analysing their nuclear, mitochondrial (mt), and chloroplast (cp) genomes using multiple molecular DNA markers. A total of 465, 446 and 462 genetic loci were produced with RFLP, RAPD and ISSR fingerprinting in the micropropagated plants and the corresponding mother plant of C. sinensis clone U (UPASI) 26, and C. assamica ssp. assamica clones U3 and U27, respectively. RFLP fingerprinting was performed using six restriction endonuclease digests and 14 mt and cp gene probes in 84 enzymeprobe combinations. For PCR fingerprinting, 50 RAPD and SSR primers were used for amplifications. The micropropagated plants of both the U3 and U27 clones revealed complete stability in the 462 and 446 genetic loci analysed. In comparison, 36 (7.7%) of the 465 loci were polymorphic among micropropagated plants of the U26 clone. The observed polymorphic loci were not restricted to a particular genome (nuclear or organellar), although a relatively low (7.43%) level of polymorphism was observed in the nuclear as compared to the mt genome (16.3%). ISSR fingerprinting (12.8%) detected more polymorphic loci than RAPD fingerprinting (4.28%). No polymorphism was observed in the cp genome of the micropropagated plants of the three tea clones. The rigorous screening of nuclear and two organellar genomes has demonstrated, for the first time, subtle genetic variation at the DNA sequence level in organized meristemderived micropropagated plants of tea. Clearly, this is another example demonstrating that organized meristem cultures are not always genetically true-to-type. The genomic changes in tea clones are genotype dependent rather than culture condition dependent.

In vitro mutagenesis and identification of mutants via ISSR in lily (Lilium longiflorum)

“…Although radiation is very effective in inducing mutation, many other factors, such as the auxin-to-cytokinin ratio and their perspective concentration, nutritional conditions and in vitro stress, may also induce somaclonal variation (Devarumath et al 2002). This may account for why four adventitious buds with mutant phenotypes were also observed in the control group, equivalent to a mutant frequency of 0.47% (Table 2).…”

Efficient long-term conservation of Taraxacum pieninicum synthetic seeds in slow growth conditions

“…In turn, high probability of genetic change occurs in callus and protoplast culture (Krishna et al 2016). However, irrespective of the source of plant material, somaclonal variation cannot be excluded, as it was demonstrated in study on Camellia sinensis and C. assamica, in which organized meristem cultures were not always genetically true-to-type (Devarumath et al 2002). Also Evans (1988) reported considerable variation in adventitious Nicotiana alata plants regenerated directly from leaf explants without a callus intermediate.…”

Genetic stability at nuclear and plastid DNA level in regenerated plants of Solanum species and hybrids

“…Similarly, Devarumath et al (2002) did not detect any polymorphism in plastid genome of the micropropagated plants of three tea clones. The chloroplast genome has been found to remain stable also in regenerants of Coffea arabica (Rani et al 2000) and F. vulgare (Bennici et al 2004).…”

“…Kuznetsova et al (2005), performing ISSR analysis on Pisum sativum regenerants, found that the percentage of polymorphism ranged from 0.7% to 15%. In Camellia somaclones, ISSR markers allowed the detection of 12% polymorphism (Devarumath et al 2002). Interestingly in S. tuberosum Sharma et al (2007) found a very low level of AFLP marker profile variation amongst plants regenerated (Bennici et al 2004).…”

“…By contrast, both genic and chromosomic mutations like inversion, deletion or translocation can be detected by molecular markers. Since mutations could occur both on nuclear DNA and on mitochondrial (mt) or chloroplast (cp) DNA (Kawata et al 1995;Rani et al 2000), various DNA markers like RAPD (random amplified polymorphic DNA), ISSR (inter simple sequence repeat) for nuclear genome screening (Gostimsky et al 2005), and RFLP (random fragment length polymorphism) for both nuclear and organellar genome screening (Devarumath et al 2002) are required to assess the genetic stability of somaclones. The occurrence of somaclonal variation is influenced by several factors such as explant type, culture medium, age of donor plant, number of subcultures and tissue culture system itself.…”