In Vitro Mass Cloning of Dianthus Chinensis – A Horticultural Important Plant

Abstract

The present investigation was carried out on a horticultural important plant Dianthus chinensis var. heddiwigii (China pink) belonging to the family Caryophyllaceae. The different vegetative plant parts viz. shoot apices, axillary buds, stem and leaves were excised from field grown healthy plants and thereafter cultured on variously supplemented Murashige and Skoog’s medium for multiple shoot proliferation, de novo adventitious shoot and root formation, callus induction and organogenesis. The leaf segments own a great organogenic potential as they exhibited a high efficiency of direct de novo adventitious root and shoot formation. Prolific root regeneration occurred from the entire surface of leaf explant on higher concentrations of NAA (2-4 mg/L) and with the incorporation of CM (15%) to NAA (2-4 mg/L) + Kn (1 mg/L) supplemented medium. The roots were thin, white and bore dense root hairs. A high efficiency of adventitious shoot formation from leaf explant was observed on MS medium supplemented with Zeatin (4 mg/L) or NAA (0.5 mg/L) + BAP (1mg/L) forming 20-25 shoots from the cut ends of leaf explant without the formation of intervening callus. In contrast, stem segments exhibited comparatively a low frequency of direct de novo adventitious shoot and root formation. Out of the various growth regulators tested, lower concentrations of IBA (0.5-1 mg/L) promoted direct rooting from the explant. Out of the variously tested cytokinins, only BAP (2 mg/L) was effective in generating shoots from the stem segment forming 20-22 shoots after 8 weeks of planting. Callusing of the leaf and stem segments occurred immediately after culturing on optimal chemical milieu. Among the various growth regulators tested, optimal callusing of the leaf explants was observed on Zeatin (2 mg/L) or TDZ (4 mg/L) or IBA (2-4 mg/L) or 2,4-D (4 mg/L) + BAP (1 mg/L) supplemented medium. Murashige and Skoog’s agar gelled medium supplemented with IBA (2-4 mg/L) or NAA (0.5-4 mg/L) or NAA (2-4 mg/L) + Kn or BAP (1 mg/L) with or without coconut milk (15%) turned out to be optimal for initiation and sustained growth of callus from the stem segments. However, MS medium supplemented with higher concentrations of IBA (2-4 mg/L) was the best medium reported for callus induction from leaf as well as stem segments. The calli thus formed were green, hard, compact and fast growing. They were heterogeneous being composed of parenchymatous ovoid, oblong, semicircular cells or those with aberrant shapes. The spectrum of induced differentiation from calli was wide and involved xylogenesis, rhizogenesis and caulogenesis. The tracheids occurred either singly or were grouped together and were mostly elongated having reticulated thickenings on their walls. Best root differentiation from leaf and stem calli occurred on MS medium supplemented with IBA (2-4 mg/L) and NAA (1and 4 mg/L) where the roots formed were thin, white having dense root hairs. Shoot differentiation from the leaf callus occurred on 2, 4-D (2-4 mg/L) + BAP (1 mg/L) either alone or in conjunction with CM(15%) forming 8-10 shoots after 8 weeks. Dianthus exhibited a high degree of propensity of multiple shoot proliferation from shoot apices and nodal segments. Multiple shoot formation was observed on MS medium supplemented with BAP or Kn or Zeatin or TDZ (1-4 mg/L) and NAA (0.5 mg/L) + BAP or Kn (1-4 mg/L). Best results were, however, obtained on BAP (4 mg/L) supplemented medium where 70-80 shoots were regenerated from single shoot apex or axillary bud. Once the clusters of shoots were formed, small clumps of 5-6 shoots were excised and transferred onto fresh multiplication medium where the increased drastically forming 140-150 shoots after 4 weeks. Regenerated shoots formed from different vegetative parts were carefully rescued from the culture vessels and transferred to root inducing media to form complete plantlets. Best rooting response (60%) was observed on BMS medium forming well developed bunch of roots at the base of the stem. The plantlets thus formed were acclimatized and attempts were made to establish regenerated plantlets into the soil.