In Vitro Propagation and Detection of Somaclonal Variation in Phalaenopsis gigantea as Affected by Chitosan and Thidiazuron Combinations

Abstract

<jats:p>Protocorm-like bodies (PLBs) multiplication is one of the most preferable in vitro methods to increase the number of orchids that produce very few seeds or seeds that are not able to germinate. In the present study the effects of chitosan and thidiazuron (TDZ) combinations on multiplication, differentiation, and genetic stability of <jats:italic>Phalaenopsis gigantea</jats:italic> PLBs were investigated using different media. Initial PLBs were cultured in solid New Dogashima (ND) medium and Vacin and Went (VW) medium supplemented with different concentrations of chitosan (0, 5, 10, 15, 20, and 25 mg·L<jats:sup>–1</jats:sup>) and TDZ (0, 0.1, and 0.5 mg·L<jats:sup>–1</jats:sup>). The highest mean number of PLBs (353 PLBs) was observed in ND medium with 10 mg·L<jats:sup>–1</jats:sup> chitosan and 0.1 mg·L<jats:sup>–1</jats:sup> TDZ combination after 20 weeks of culture. Some PLBs differentiated into mature PLBs with a profusion of leaves on the apical region, and tiny plantlets started to develop after 10 weeks of culture. The highest mean number of shoots was observed in VW supplemented with 10 mg·L<jats:sup>–1</jats:sup> chitosan and 0.5 mg·L<jats:sup>–1</jats:sup> TDZ (16 shoots). Intersimple sequence repeat (ISSR) markers were used to determine the genetic fidelity among mother plant and PLBs obtained from each subculture stage of solid ND medium supplemented with 10 mg·L<jats:sup>–1</jats:sup> chitosan and 0.1 mg·L<jats:sup>–1</jats:sup> TDZ (the optimal treatment for PLB proliferation). Dissimilarity of 5% occurred between the mother plant and PLBs obtained after 16 weeks of culture. The range in the similarity coefficient varied from 0.80 to 1.0, and only 20% dissimilarity occurred between mother plant and PLBs after 20 weeks of culture.</jats:p>

Clonal Fidelity of Micro-propagated Phalaenopsis Plantlets Based on Assessment Using Eighteen Ph-Pto SNAP Marker Loci

“…In this research, the presence of 11.8 % plantlet variants was reported based on SNAP marker analysis. Although gene-specific and co-dominant SNAP marker based on nucleotide sequence variations of Pto gene were used, this finding was in line with the previous study using genome-wide dominant markers of either RAPD or ISSR (Khoddamzadeh et al, 2010;Samarfard et al, 2014).…”

“…The result in this study was in line with that of Chen, Chen, & Chang (2002) which showed supplementation of TDZ (0.1 -1.0 mg L -1 ) to the PLB proliferation medium did not significantly affect the proliferation rate and the PLBs numbers on Epidendrum. In different circumstances, Samarfard et al (2014) showed that the addition of 0.1 mg L-1 TDZ significantly increased proliferation of Phal. gigantea PLBs than that of 0.5 mg L -1 TDZ in the PLB proliferation stage.…”

“…However, they detected 17 % in RAPD marker profile variations among the proliferated, six months old culture of PLBs. In various elucidation using ISSR markers, Samarfard et al (2014) reported the presence of 20 % in ISSR marker profile variations in Phal. gigantea among the mother plant and the 20 weeks old culture of secondary PLB.…”

“…Therefore, the preferred orchid tissue culture protocols should support the increase in PLBs multiplication, total yield, and plantlet conversions without inducing somaclonal variations (Pornpienpakdee et al, 2010). Samarfard et al (2014) found that a medium supplemented with a combination of 0.1 mg TDZ and 10 mg L -1 chitosan gave the lowest frequency of somaclonal variation on Phalaenopsis gigantea micropropagation.…”

An Overview of Orchid Protocorm-Like Bodies: Mass Propagation, Biotechnology, Molecular Aspects, and Breeding

“…Ref. [92] reported increases in SV after the third subcultures of PLBs in proliferation medium (NDM + 0.1 mg L −1 TDZ and 10 mg L −1 chitosan) with same ISSR profile until third subculture, 95% at fourth and 80% at fifth subculture of PLBs.…”