Callus Callus- a mass of undifferentiated cells produced

at wound edge.

Can be grown in vitro and induced to differentiate by varying the ratio of hormone auxin and cytokinin in the medium.

Callus culture can be produced from any part of the plant eg; root, shoot, leaves, leaf base meristems, mature and mature embryos, etc.

Embrogenic callus – callus cultures under suitable conditions are capable of producing embryos (young plants).

Plant regeneration through somatic embryogenesis

Somatic embryogenesis (SE) is the process by which somatic cells, under induction conditions, generate embryogenic cell, which go through a series of morphological and biochemical changes that result in the formation of somatic embryos.

SE has potentially biotechnological applications such as artificial seeds, micropropagation, transgenic plants, etc.

The objective of the present study is to identify a suitable medium for callus induction and the ability of plant regeneration through somatic embryogenesis.

Materials and methods Sterilization of seeds

Mature seeds of rice cultivar Fujisaka 5 used were obtained from MARDI, Seberang Perai, Penang. The seeds were dehusked and sterilized with 70 % alcohol for 10 min followed by 30 min sterilization in 20 % Clorox® added with a few drop of Teepol.

After rinsing several times in sterile distilled water, the seeds were inoculated on MS (Murashige & Skoog, 1962) supplemented with 2.0 mg/L 2,4-D.

The cultures were placed in a culture room with a

temperature of 25 ± 2ºC.

The growth and contamination rate were observed every week.

Callus production (mg ± s.d) from different explants of Oryza sativa L. cv

Fujisaka 5 on MS supplemented with 2,4-D (0-10 mg/L) after 4 week of culture

Explants typeExplants type 2,4-D (mg/L)2,4-D (mg/L)

00 22 44 66 88 1010

Mature seedsMature seedsRootsRootsLeavesLeaves

------

40 ± 2.240 ± 2.260 ± 4.860 ± 4.8

--

26 ± 626 ± 636 ± 2.436 ± 2.4

--

35 ± 2.535 ± 2.533 ± 2.833 ± 2.8

--

33 ± 1.233 ± 1.269 ± 3.669 ± 3.6

--

36 ± 1.836 ± 1.826 ± 2.826 ± 2.8

--

Root explants showed to be the best explant for callus forming ability in term of weight.

But the callus formed are highly unorganized, yellowish white in colour and differentiated into roots.

Two type of callus, embryogenic and non embryogenic were obtained from the embryo scutellum. The embryogenic callus were relatively dry, yellowish in colour, compact and nodular in appearance.

The leave explants were found to be not suitable as the explant source for callus induction

Callus Induction

Callus induction from (A) mature seed (B) root segments of Oryza sativa L. cv. Fujisaka 5 after 4 weeks of culture; (C) Fine root hairs formed on the surface of the root-derived callus after three subcultures

Mature seed were selected as the best explant for callus induction of rice.

A B C

Plant regeneration

To study the effect of 2,4-D and kinetin combination, approximately 0.5 g of embryogenic callus of Fujisaka 5 were inoculated onto MS medium supplemented with different concentration of 2,4-D (0, 0.5 or 1.0 mg/L) and kinetin (0, 0.5 or 1.0 mg/L) and maintained under continuous light .

Observations on callus structure and colour were made every week and data were taken after 3 weeks of culture.

the callus were subcultured onto MS PGR-free medium plant regeneration.

Plant Regeneration After three weeks of

culture in the MS medium containing 2,4-D and kinetin, the biomass of the embryogenic callus increase in all the MS medium containing (0.5 – 1.0 mg/L) 2,4-D and kinetin.

The embryogenic callus remained yellowish in color.

Effect of 2,4-D and kinetin supplemented into MS on proliferation of plant Fujisaka 5 embryogenic callus. embryogenic callus.

0.000

0.500

1.000

1.500

2.000

2.500

0 0.5 D 1.0 D 0.5 K 0.5 D0.5 K

1.0 D0.5 K

1.0 K 0.5 D1.0 K

1.0 D1.0 K

Control

Medium

Fre

sh w

eig

ht

(g)

But after subculture the embryogenic callus onto MS basal medium, there were some green embryos detected from all medium and only 1 to 2 plantlets/bottle regenerated from these green embryos.

This might indicate that higher concentration of 2,4-D or kinetin were needed for plant regeneration.

Plant regeneration of rice cv. Fujisaka 5

Somatic embryos and complete plant of rice cv. Fujisaka 5.

Histological studyCallus tissues were removed from the medium and fix in

FAA solution.

After dehydration through a gradual ethanol siries, the samples were embedded in xylene wax and cut into 10 um thick sections.

The samples were stained with safranin and fast green.

Culture were examined and photographed with a stereo zoom microscope (SZH, Olympus).

Histological observation

Histological study showing the somatic embryos differentiated into young primordia leaves

Conclusion Embryogenic callus could be induced from

the mature seeds of O. sativa L. cv. Fujisaka 5 on MS medium supplemented with 2.0 mg/L 2,4-D.

The somatic embryos can be generated into plantlets when they were transferred onto MS basic medium.