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Organogenesis (shoot)

12.6 (±3.1)

aMean of 10 replications

Supplemented with 3 mgl"1 2,4-D.

Supplemented with 0.2 mgl"1 2,4-D and 10 mgl"1 BAP.

aMean of 10 replications

Supplemented with 3 mgl"1 2,4-D.

Supplemented with 0.2 mgl"1 2,4-D and 10 mgl"1 BAP.

6 to 8 months, whereas while it takes about 5 to 7 months for the vegetative bud-derived callus.

In callus derived from ovary tissues morphogenesis was observed on MS media supplemented with 5 to 10 mgl-1 BAP or kinetin and 0.1 to 0.2 mgl-1 2,4-D and also with 5 mgl-1 BAP or kinetin and 0.2 mgl-1 2,4-D after four to five subcultures. In both the media white globular heart-shaped embryo-like structures were observed in 67 to 80 percent of the cultures. In about 22 percent of the cultures both organogenesis and embryogenesis were observed in the same culture. This process continued in the subsequent cultures on the same medium. The embryogenic calli were separated and used for subsequent cultures. When growth regulators were removed from the culture medium at this stage, the callus turned green and the rate of morphogenesis increased considerably, resulting in many embryo-like structures that could be separated easily with a gentle tap. On subsequent transfer to fresh medium without growth regulators, the embryoids developed into complete plants. The somatic embryos produced secondary embryoids by adventitious embryogenesis resulting in a large number (100 to 300) of tiny embryoids (see Figure 4.1e). This type of multiplication continued on growth regulator-free medium even after 2 years of continuous culture, indicating that once the callus turns embryogenic, growth regulators are not needed for further multiplication. The embryoids produced were either compactly arranged or loosely arranged. The development of these embryoids into complete plantlets was higher when NAA (1 mgl-1) was added to the culture medium, which also enhanced rooting. This protocol with a production potential of a large number of tiny propagules is ideally suited for in vitro manipulations such as in vitro mutagenesis, in vitro polyploidization, and in vitro selection against biotic and abiotic stresses. The somatic embryos are ideally suited for direct DNA transfer using a particle-delivery system and development of transgenics.

The explants differed in their morphogenetic response with regard to the morphoge-netic pathway as well as the plantlets regenerated. The plant regeneration was by organogenesis in leaf- and anther-derived callus, whereas it was by both organogenesis and embryogenesis in vegetative bud-derived and ovary-derived callus. Histological studies have proved the origin of both organogenesis and embryogenesis. Embryo development followed typical monocotyledonous stages of development from globular, heart-shaped to torpedo-shaped embryos with clear-cut scutellum and coleoptile development. The embryo showed clear shoot and root poles with no vascular connection between the host tissue and the embryo. The explants differed with respect to days taken for morphogenesis and their plant regeneration potential. Anther-derived callus was quickest to respond (120 days) compared to leaf-derived callus, which is slowest (162 days). The mean number of plantlets recovered ranged from 12.6 from anther-derived calli to 47.6 in ovary-derived calli (see Table 4.2). The embryogenic calli derived from ovary were canalized and produced an increasing number of somatic embryos in the subsequent cultures. These somatic embryos also resulted in repetitive embryogenesis and budding resulting in a large number (over 100) of somatic embryos in a given culture tube. Thus, the ovary was the best among the explants tried for plant regeneration and recovery. This efficient plant-regeneration system is ideally suited for in vitro selection, in vitro mutagenesis, and genetic manipulation experiments (Nirmal Babu et al., 1996b).

Shoots developed from the callus were placed on the modified MS medium with 1 mgl-1 of NAA for rooting that was earlier identified as ideal for root induction. An extensive root system developed within 5 weeks in this medium. Rooting was better when liquid medium was used instead of solid medium.

Successful plant regeneration in ginger was earlier reported (Nadgauda et al., 1980; Ilahi and Jabeen, 1987; Kulkarni et al., 1984; Malamug et al., 1991; Kackar et al., 1993). The explants used were young sprouts and young leaf segments and the plant regeneration was via organogenesis and embryogenesis. Earlier studies indicate BAP at high concentration resulted in morphogenesis and plant regeneration from these explants.

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