Protocols for Micropropagation of Woody Trees and Fruits

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V.M. KULKARNI ET AL.
2.1.3. Greenhouse Hardening of Plantlets
After 3–4 weeks, plantlets with well developed root-system (Figure 2E) are carefully
removed from the culture vessel and gently washed in running tap water to remove the
entire gelled medium. If needed, a paint brush with smooth bristles can be used to
remove adhering gel pieces. The plantlets shall then be transferred to perforated
polythene bags, portrays or suitable containers filled with autoclaved mixture of soil
and a suitable commercial hardening mixture such as Soilrite, Cocopeat etc. (Figure
2F). The plantlets are maintained in the greenhouse for 2–4 months (depending on
the growth), under natural light with relative humidity of 90–100% and an ambient
temperature of 25°C. Approximately 40–60 cm tall plants can be field planted for
evaluation.
2.2. Somatic Embryogenesis Pathway
The development of embryogenic cell suspensions (ECS) capable of producing
embryos and plants is achieved using different types of explants. In majority of the
reports, the immature male flowers and/or shoot-tip derived scalps are a choice
system for developing ECS cultures. In both these methods, at least a few hundred
explants are required to be cultured and maintained for several months to be able to
obtain good quality embryogenic callus. Although somatic embryogenesis in banana
is now a well-established method (Ganapathi et al., 2002; Strosse et al., 2003), the
initiation of a “genotype-independent” embryogenic cell culture is still far from
routine. This is mainly due to the low embryogenic potential, the long gestation
periods required for initiating an embryogenic cell suspension, the risk of somaclonal
variation (in case of long culture periods) and contamination problems. The
detailed protocols for obtaining embryogenic callus, suitable for initiation of
superior quality ECS, are given below.
2.2.1. Initiation of Embryogenic Callus Cultures through Immature Male
Inflorescence Culture
The male inflorescences are collected from field growing plants after complete
bunch emergence (Figure 3A) and washed well with distilled water. These are
trimmed to 10 cm in length (Figure 3B), and can be kept until sterilization under a
laminar flow in a beaker with a few drops of sterile water and covered with
aluminium foil. These are surface-sterilized in 70% ethyl alcohol for 2 min and the
immature male flower buds are isolated using a binocular dissection microscope.
The immature flowers are isolated from position 16 to 1 (1 being the innermost
flower bud cluster, closest to the floral tip). The innermost whorl at the tip can be
cultured as such. The excision and isolation should be done with a sharp and pointed
surgical blade. While culturing, it must be observed that the cut surface is in contact
with the callus induction medium (BM1, Figure 3C) in 9 cm Petri dishes and
incubated in total darkness under standard tissue culture conditions.