Protocols for Micropropagation of Woody Trees and Fruits

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M. MISHRA ET AL.
micropropagation. Most of the reports on papaya micropropagation using mature
explants are therefore conflicting. Somatic embryogenesis system using hypocotyl
(Fitch, 1993) or immature zygotic embryos (Fitch & Manshardt, 1990) has been
successful in vitro regeneration.
The micropropagation protocol described herein is based on direct somatic
embryogenesis using immature zygotic embryo for somatic embryo induction and
subsequent plant regeneration.
2.1. Sterilization and Explant Preparation
2.
Select 90- to 120-day-old immature green papaya fruit from the mid position of fruit
bunch. Wash it in the running tap water. Surface sterilize the fruit with 1.05%
sodium hypo chloride (NaOCl) solution containing 1 drop of Tween 20 for 1 h and
wash with autoclaved distilled water five times. Bisect the fruit under aseptic
condition and then scoop out white, plump immature seeds. With forceps and scalpel
remove the testa, cut it from one side and press the immature seed from mid portion
to take out immature zygotic embryos (Figure 1).
Figure 1. Immature zygotic embryos of papaya. Left a group of embryos, right a single embryo.
2.2. Culture Medium
EXPERIMENTAL PROTOCOL
2.2.1. Culture Medium Preparation
Either commercially available pre-packaged MS (Murashige and Skoog, 1962) salt
formulation or in-house prepared stock solutions can be used for culture medium
preparation. Table 1 lists the ingredients of MS medium and specifies other
inorganic and organic additives. Appropriate amount of stock solution of salts and
plant growth regulators should be transferred to a one-litre flask and made up
volume to one litre (Table 1). The medium is solidified with 8 gm/l agar. Prepare
half strength MS basal medium containing all additives except plant growth
regulators, which are added according to culture stage as shown in Table 1. Adjust
pH of the medium to 5.7 with 1M NaOH and HCL and dispense in culture vessels
and autoclave them for 20 min at 121°C at 20 kg cm –2 pressure. Autoclavable
polycarbonate or glass transparent bottles are suitable for papaya tissue culture
work. Inoculate tissue after 48 h in the medium to ensure contamination free
conditions.