Plant Regeneration

Two-month-old calli derived from various explants—vegetative bud, young leaf, ovary and anther tissues—were cultured on a series of MS basal media supplemented with varying levels of auxin (2,4-D) and cytokinins (Kinetin and BAP) for morphogenesis and plant regeneration (Table 4.3)

When fresh calli, immediately after its induction, was cultured on MS medium without growth regulators, it resulted in rhizogenesis. The cytokinins, kinetin, and BAP when used individually did not result in organogenesis even after four to five cycles of subculture. But addition of cytokinins (kinetin and BAP) at concentrations of 5 to 10 mgl"1 in the presence of 0.2 mgl"1 2,4-D resulted in induction of morphogenesis—both organogenesis and embryogenesis—and subsequent development of plantlets. MS medium supplemented with 10 mgl"1 BAP and 0.2 mgl"1 2,4-D gave the best mor-phogenic response. Once the morphogenic pathway of the cultures was determined, they continued to show their morphogenic potential in subsequent subcultures even after 2 to 3 years.

In vegetative bud-derived callus, the number of plantlets developed per culture were higher in BAP (28 to 60) than in kinetin (8 to 26). The rate of shoot production and plantlet formation increased considerably to 36 to 62 shoots per culture when growth regulators were completely excluded from the medium in later subcultures after the induction of morphogenesis (see Figure 4.1c).

MS medium supplemented with 10 mgl"1 BAP and 0.2 mgl"1 2,4-D was also the best for leaf-derived callus. The number of shoots per culture was higher in BAP (15 to 35). Transfer to growth regulator-free culture medium after initial morphogenesis resulted in enhancement of plant regeneration to 30 to 60 shoots. The time taken for the leaf-derived callus to differentiate and develop into a complete plantlet ranges from

Table 4.3 Effect of explant on plant regeneration through callus phase on MS basal mediuma

Sl. No.


Callus production (g)b

Days taken for morphogenesis'

Morphogenetic pathwayb

No. of plantlets ±SDb

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