Coconut Callus Initiation for Cell Suspension Culture

Abstract

The development of a cell suspension culture system for the scaling up of coconut embryogenic callus (EC) production would drastically improve efforts to achieve the large-scale production of high-quality clonal plantlets. To date, the hard nature of coconut EC appeared to be the main constraint for developing cell suspension cultures. Hence, this study attempted to acquire friable EC through the following approaches: The manipulation of (1) medium type and subculture frequency, (2) a reduced 2,4-dichlorophenoxy acetic acid concentration during subculture, (3) the nitrate level and the ammonium-to-nitrate ratio, and the addition of amino acid mixture, (4) the addition of L-proline, and (5) the reduction of medium nutrients. Unfortunately, none of these culture conditions produced friable coconut EC. Even though friable EC was not achieved via these approaches, some of the conditions were found to influence the formation of compact EC, therefore these results are important for further studies focused on somatic embryogenesis in coconut and other species.

Keywords: Cocos nucifera L.; callus; cell suspension culture; coconut; embryogenic callus; somatic embryogenesis; tissue culture.

Figure 1

The type of compact callus formed. (A) Embryogenic callus (EC). (B) Non-EC. (C) Non-embryogenic callus appearing black in colour due to a coating of activated charcoal and produced from liquid medium.

Figure 2

The effect of different medium types with various subculture frequencies (Control, T1 to T5, see Section 4.4) on the percentage of callus and compact embryogenic callus formed. The mean ± standard error for different treatments in each bar followed by different letters was significantly different for that parameter (Tukey’s test, p ≤ 0.05); n = 5.

Figure 3

(A) Compact embryogenic callus (EC) used in the study to form friable EC. (B) Mature somatic embryos and (C) roots forming on compact EC after two subculture cycles.

Figure 4

The development of Malayan Yellow Dwarf plantlets after (A) 4, (B) 5, (C) 7, and (D) 8 months on a plant growth regulator-free Y3 medium containing activated charcoal.

Figure 5

The formation of compact embryogenic callus for (A) control and treatments (B) T1, (C) T2, (D) T3, (E) T4, and (F) T5 (Control, T1 to T5; see table in Section 4.6) after 3 months.

Figure 6

The effect of various combinations of nitrate and ammonium ion concentrations placed into the Y3 medium with and without amino acids (Control, T1 to T5; see table in Section 4.6) on the percentage of callus and compact embryogenic callus formation. Each bar represents the mean ± standard error; n = 5. Bars covered by the same letter were not significantly different (Tukey’s test, p ≤ 0.05).

Figure 7

The effect of reduced nutrients (Control, T1, and T2) on the percentage of compact embryogenic callus (EC) formed and explants with necrosing (Browning) tissues. Mean value ± standard error is presented on each bar. The mean value of each treatment for each coconut variety followed by different letter was significantly different (Tukey’s test, p ≤ 0.05); n = 3. Control: Eeuwens Y3 medium; T1: Modified Eeuwens Y3 medium with only 10% N, P, and K and 10 mg L⁻¹ thiamine hydrochloride; T2: Gresshoff and Doy (GD) medium.

Figure 8

The effect of reduced nutrient concentration on the formation of compact embryogenic callus (EC), non-embryogenic callus (non-EC), and browning callus for the variety Indian Yellow Dwarf.

Figure 9

The effect of reduced nutrient concentration on the formation of compact embryogenic callus (EC), non-embryogenic callus (non-EC), and browning callus for the variety Sampoorna.

Figure 10

Histological section of compact embryogenic callus derived from a plumule obtained from a CAIRNS embryo, and after periodic acid Schiff’s staining. Nuclei were stained blue, while starch and polysaccharidic sheath (PS) were stained pink or purple. Numerous starch (S) granules are observed surrounding the large nucleus (N) with visible nucleolus (Nu).

Figure 11

Histological sections of non-embryogenic callus stained with periodic acid Schiff’s reagent. Parenchyma (Pa) cells were observed in the central zone (A) at a magnification of 200 µm. No starch granules or meristematic cells were observed, under high resolution (B), but nuclei (N) were visible.