Cold treatment prompts embryogenic callus and cytodifferentiation in the anthers of tea (Camellia sp.) genotypes
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The simple and rapid protocol was developed to induce androgenic embryoids from immature anthers of tea genotypes. Anthers from unopened flower buds of ~ 5 to 6 mm size were subjected to two temperature treatments at 4 °C and 25 °C temperatures for 5 days and cultured onto two mediums, half strength MS liquid and 0.8% agar medium. The inoculated cultures were incubated under complete dark (24 h) and photoperiod (9:15 h) conditions. Anthers of nine tea clones (UPASI-1, UPASI-2, UPASI-3, UPASI-8, UPASI-10, UPASI-20, UPASI-28, and certain estate selections, ATK-1 and CR-6017) initially cultured onto half strength Murashige and Skoog’s (MS) liquid medium supplemented with 2,4-dichlorophenoxy acetic acid (9.05 µM 2,4-D) combined with kinetin (2.44 µM Kn) and 6-benzylamino purine (2.22 µM BAP). The pre-treated anthers at 4 °C for 5 days of genotype UPASI-3 induced significantly higher percentage of callus induction when cultured onto half strength MS agar medium under both continuous dark and photoperiod (9:15 h) conditions. Percent of callus production was significantly (P < 0.001) influenced by the genotypes (G) and plant growth regulators (P) while it was not affected by culture conditions (Dark; D/Photoperiod: L). Irrespective of genotypes studied, medium supplemented with BAP is superior to Kn in inducing androgenic potential and promotes the emergence of embryoid structures in a short time span of 45 days and the emergence of morphologically prominent globular mature embryoid callus was identified for 55 days and subsequent cytodifferentiation were achieved only in medium fortified with 2,4-D along with BAP. The results found that cold treatment and culture medium accelerated the development of embryogenic calli. Histological evidences of embryoids revealed that in vitro cytodifferentiation rendering to cellular totipotency developed different meristematic cells and vascular elements. Anthers provided with cold treatment induced callus in the liquid growth medium. The liquid growth medium formulation used here is suitable for inducing androgenic potential and promoted callus formation from anthers in less time span for 10–15 days. Callus maturation and immature embryoid callus was induced for 45 days and morphologically prominent round mature embryoids were appeared by 55 days. And the histological studies reveal the trend of in vivo cytodifferentiation rendering to cellular totipotency while growing in vitro. Our experiment concludes the profuse callus induction and embryoid induction by using liquid growth medium formulation, which is difficult task in anther culture of tea because of its highly recalcitrant nature. Liquid medium hastens the androgenesis process in tea anthers and induced embryoids when cultured onto same solid agar medium.
KeywordsAndrogenesis Embryoids Histology Procambium Uninucleate microspores Scalariform xylem vessels Protoxylem Metaxylem
2,4-Dichlorophenoxy acetic acid
Plant growth regulators
Tetramethyl rhodamine isothiocyanate
BAP liquid formulation
Kinetin liquid medium formulation
The authors are thankful to the National Tea Research Foundation (NTRF), Government of India, Kolkata for the financial support in the form of research Grant (NTRF: 158/2012) to conduct this study. The authors are also grateful to Dr. B. Radhakrishnan, Director, UPASI TRF TRI, and Valparai for his constant support.
NPK designed and executed the work. NPK and ERE prepared the manuscript. CKN and MKP supervised the experiments, RKR helped in editing the manuscript. All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
This article contains original research carried out at Plant Physiology and Biotechnology Division, UPASI TRF TRI, Valparai. All the authors declare that they have no conflict of interest.
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