Protocols for Micropropagation of Woody Trees and Fruits

2.4. Culture Conditions
MICROGRAFTING IN GRAPEVINE 255
All media should be solidified with gelrite (Sigma, 3 g l –1 ) and pH should be
adjusted to 5.8 prior to autoclaving at 121°C, 103.4 kPa pressure, for 20 min.
Preferably 290 ml clear wide-mouth disposable tissue culture tubs with snap-on lids
containing 50 ml medium are the culture vessels ideal and desirable for such work.
The wide mouth allows easy access during the in vitro grafting of scion on to the
rootstock and this type of vessel generates good air circulation needed for the
stability of the graft. Cultures should be placed under 16 h light, 8 h dark photoperiod
at 30 µmol m –2 s –1 light intensity at 24°C (Pathirana & McKenzie, 2005).
2.5. Rooting and Transfer to Soil
2.5.1. Rooting of Shoots
Rooting in in vitro grafted plants is achieved on defined medium (Table 1a) for a
month, after which the grafted plants are ready for transplanting. Some rootstocks do
not respond to this medium. In such cases it is necessary to achieve rooting of the
rootstock prior to the micrografting step, on medium ‘e’ (Table 1) for 6 weeks. The
roots are trimmed and the young plants are transferred to sterile soil mix. Air
circulation is gradually increased and the plants are ready for the greenhouse
transplantation. Rooting of grafted plants can also be done as a two step procedure
in which roots are only first initiated in vitro and subsequently grown in vivo on root
growing media such as peat, perlite or vermiculite (or a mixture of all three), which
are commercially available (Sigma-Aldrich or at local nurseries).
2.5.2. Acclimatization of Micrografted Plants
The successful grafts require appropriate acclimatization to external environmental
conditions in order to obtain a high percentage of survival. It is necessary to have
minimum 2–3 leaves developed on the grafted plant before transfer to soil. The time
required to reach this stage varies between 5 and 6 weeks after the graft is stabilized.
Steam sterilized soilmix suitable to grow grape is always recommended. The plants
should be covered with polythene bags with perforations, to retain appropriate
moisture and humidity and should be kept in rooms equipped with controlled
conditions of temperature, light and humidity.
2.5.3. Graft Incompatibility and Histology
The term ‘incompatibility’ with respect to grafted fruit trees is defined as a phenomenon
of premature senescence caused by physiological and biochemical factors
(Feucht, 1988). When two plants of different genotype are grafted, the degree of
adaptation or ageing depends on the plasticity of both partners to perform in the
enforced symbiosis. Hence, diseases (causative agents), genotypic response of the
symbiont to the changed environment or severe environmental stress may shorten
the life span of the sensitized graft combination. Histological observations in
Opuntia spp., micrografts have revealed that there are five developmental stages of
graft union formation (a) development of a necrotic layer, (b) proliferation of a