Protocols for Micropropagation of Woody Trees and Fruits

2.5. Rooting and Acclimatization
MICROPROPAGATION OF BLACK LOCUST 197
The shoots approximately 2-cm long were transferred on the rooting medium (Table 1,
Figure 1E). Four shoots were placed in each vessel (250 cm 3 in size) and usually the
roots protruded after 21 days of culture on the rooting medium. At this stage, plantlets
had dark green leaflets and 2 to 3 white roots.
Since in vitro plantlets are grown under highly controlled culture conditions, they
would desiccate and die by transferring them directly to the field conditions. There-
fore, the plantlets were acclimatized at first in the greenhouse conditions for 21
days. The culture vessels were placed facing direct sunlight in order to expose them
to the scattered light and daily temperature fluctuations.
Acclimatization of the plantlets to the field conditions took more than 10 days.
An adaptation period of less than 10 days was not sufficient and a long adaptation
period (30 days) might cause nutrient deprivation in the medium. Moreover, roots
were still growing and extending for a long period of time in the rooting medium.
These roots were easily damaged while cleaning them with water for removing agar
sticking to them and consequently decreased in plantlet survival rate.
In order to avoid dehydration and burning, the tender plantlets were kept away
from the strong light conditions. Adaptation to sunshine was performed in two steps:
Firstly, the plantlets were placed under a gentle and scattered sunlight environment
to mimic the in vitro condition. Secondly, the plantlets were moved to a scattered
sunlight gradually so that they could adapt to natural growing conditions.
2.6. Transplantation
Acclimated plantlets, rooted in the culture vessels, were transplanted in a rooting
mixture (sand : peat : perlite or vermiculite) for field transplantation (Figure 1E). At first,
the roots of in vitro plantlets, sticking with agar, were submerged in hot water (at
40°C) for about 30 min to wash it off. Thereafter, plantlets were transferred either
directly in sand [sand, peat and perlite (1:1:1 v/v/v)] and vermiculite mixture (1:1
v/v) or firstly in vermiculite for 21 days followed by planting in sand (see above)
and vermiculite mixture. The vermiculite was sterilized by spraying 0.3% KMnO4
solution, and it was ready to use after completely draining out KMnO4 solution.
After being adequately watered, the plants were kept in the greenhouse for about 21
days before moving them to the fields.
The selection of the rooting substrate for transplanting the plantlets should be
critically made. Vermiculite would be a better choice. The plantlets growing in it
showed vigorous and strong growth potential. Leaves were green and glossy. New
shoots developed and grew 1–2 cm long within 21 days after transplantation in the
field. On the contrary, the plantlets in perlite were stagnant and weak.
The young plants had to be transplanted to bigger containers or fields after 21
days. After that, the plants became much stronger. Their stems lignified and their
resistance to environmental stresses increased. The final survival rate of the plants
was over 98%. If the plantlets did not form roots or their roots were damaged, new
–1
roots could be induced by 500 mg L indole-3-butyric acid (IBA) solution treatment
for 5 s, before transferring in sterilized vermiculite.