In Vitro Regeneration in Cucumis melo cv. Pusa Madhuras

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Cucurbit Genetics Cooperative Report 15:62-64 (article 23) 1992

Jaagrati Jain and T.A. More
Division of Vegetable Crops, Indian Agricultural Research Institute, New Delhi – 110012, India

Introduction. Most of the cultivated forms of Cucumis melo L., such as Pusa Madhuras, are highly susceptible to field conditions at young stage of vegetative growth. Field conditions, namely the biotic factors like Fusarium wilt caused by Fusarium solani and cucumber green mottle mosaic virus (CGMMV) are responsible for eradication of cultivated forms (1, 2). Thus, it becomes essential to incorporate resistance in cultivated forms through non-conventional means of somaclonal variation, somatic hybridization, or protoplast fusion. Conventional means of interspecific hybridization has strong barriers (3).

Present study is a step towards standardization of tissue culture cycle of various explants of Pusa Madhuras and regeneration of plants in vitro, followed by screening under field conditions against CGMMV.

Materials and Methods. In Pusa Madhuras of Cucumis melo L., seeds were surface sterilized with 0.1% mercuric chloride for 7 min. and were germinated under sterile conditions. Explants such as cotyledonary leaves and epicotyl were excised from 9-day-old seedlings and inoculated onto two kinds of basal media such as Nitsch and Mirashige and Skoog. De-differentiation medium and differentiation medium were standardized for various hormonal combinations and concentrations. Regenerated plants were screened for CGMMV resistance under natural field conditions.

Results. De-Differentiation: Two kinds of explants namely epicotyl and cotyledonary leaves were tried for differentiation. Epicotyl explants with an apical meristem lead to direct regeneration of shoot. Cotyledonary explants exhibit callus induced regeneration of shoot buds and axillary bud induced direct regeneration.

Basal medium, Nitsch, had no effect on epicotyl explants but induced 49.01 + 3.40% (Table 1) of direct regeneration of shoot(s) and roots in cotyledonary leaves explants.

Murashige and Skoog (MS) basal medium favored 92.00 + 2.10% callus formation in epicotyl explants. Addition of benzyl amino-purine (BAP) 0.5 mg/l induced shoot buds in 40.47 + 4.12% of calli and favored callus formation in 66.66 + 22.05% of cotyledonary leaf explants. Addition of GA3 (0.5 mg/l) to MS basal medium also favored callus induced shoot buds formation in 46.66 + 5.77 percent of epicotyl explants. Addition of zeatin (0.5 mg/l) to MS favored root formation in both epicotyl and cotyledonary explants.

Differentiation medium: Addition of GA3 , (0.5 mg/l) to MS medium induced 41.66 + 7.21% (Table 2) of shoot buds in MS + 0.5 mg/l BAP induced callus. Addition of zeatin (0.5 mg/l) to MS medium (MBZ) had regenerative effect on MB obtained cotyledonary leaf explant callus. Regeneration of shoot buds was of the order of 0.84 + 0.25% of callus induced shoot buds (somatic embryoids), 4.20 + 1.47 percent of direct initiation of shoot (s), 10.08 + 2.50% of initiation of leaves and shoot elongation.

On the other hand, addition of IA (1 mg/l) + Kinetin (5 mg/l) to MS medium (MIK) had induction of direct shoot (s) and root(s) in 66.66 + 7.21% if epicotyl explants and 16.66 + 8.86 percent of cotyledonary leaf explants.

In conclusion, hormones such as IAA (1.0 mg/l) + Kinetin (5.0 mg/l) with MS basal medium are highly effective in inducing regeneration of shoot buds in epicotyl explant callus obtained on MS + BAP (0.5 mg/l) medium, whereas GA3 (0.5 mg/l) with MS basal medium is effective for inducing regeneration in cotyledonary leaf explant callus obtained on MS + BAP (0.5 mg/l) medium (Table 3).

Table 1. Effect of various hormones on de-differentiation and differentiation of various explants of Cucumis melo cv. Pusa Madhuras

Callus + Regeneration

Direct regeneration

Basal Media

Hormonal Supplement

Explant
Callus Formation (%)
Callus & Shoot Buds (%)
Callus + Root (%)
Initiation of Root (%)
Iniation of Shoot & Root (%)
Nitsch – Epicot. 0 0 0 0 0
Cot. leaf 15.68 + 3.39 0 0 19.60 + 3.39 49.01 + 3.40
MS – Epicot. 92.00 + 2.10 0 0 0 0
Cot. leaf 18.36 + 1.32 0 0 0 0
MS BAP (0.5 mg/l) Epicot. 9.52 + 4.12 40.47 + 4.12 0 0 0
Cot. leaf 66.66 + 22.05 0 0 0 0
MS GA3 (0.5 mg/l) Epicot. 0 46.66 +5.77 0 0 0
Cot. leaf 0 0 0 0 0
MS Zeatin (0.5 mg/l) Epicot. 0 0 7.1 + 0.0 46.42 + 3.21 0
Cot. leaf 0 0 0 55.55 + 3.21 0

Table 2. Effect of various hormones on differentiation of MB (MB + 0.5 mg/l BAP) generated of various explants of Pusa Madhuras.

De-Differentiation Medium

Differentiation Medium

Explant

No Change

Callus + Shoot Buds

Callus + Shoot

Callus + Root

Shoot Buds + Root

Shoot Elongation + Leaf Formation
MS + BAP (0.5 mg/l) (MB) MS + GA3 (0.5 mg/l) Epicot. 75.00 + 35.35 0 0 0 0 0
(MGA3) Cot. leaf 58.33 + 7.21 41.66 + 7.21 0 0 0 0
MS + Zeatin + BAP (0.5 mg/l) (0.5 mg/l) Epicot. 37.30 + 1.06 0 0 0 0 0
(MBZ) Cot. leaf 5.88 + 1.41 0.84 + 0.25 4.20 + 1.47 0 10.8 + 2.50
MS + IAA + Kinetin (1 mg/l) (5 mg/l) Epicot. 3.33 + 7.21 0 0 0 66.66 + 7.21 0
MIK Cot. leaf 83.33 + 28.86 0 0 0 16.66 + 8.86 0

Table 3. Summation of effect of hormones on differentiation and de-differentiation in Pusa Madhuras.

De-Differentiation Medium

Differentiation Medium (mg/l)

Explant

Regeneration (%)

Callus Formation (%)

No Response (%)
MS + BAP (0.5 mg/l) – Epicot. 40.47 + 4.12 9.52 + 4.12 50.00
Cot. leaf 0 66.66 + 22.05 33.33
MS + GA30.5 mg/l0 – Epicot. 0 46.66 + 5.77 53.84
Cot. leaf. 0 0 0
MS + Zeatin (0.5 mg/l) – Epicot. *46.42 + 3.21 (Rooting) 0 46.43
Cot. leaf *55.55 + 3.21 (Rooting) 0 44.44
MB + MGA3 (0.5) Epicot. 0 75.00 + 35.35 25.00
Cot. leaf *41.66 + 7.21 58.33 + 7.21 0
MB -> MB + BA + Zea (0.5) (0.5) Epicot. 0 37.30 + 1.06 62.69
Cot. Leaf 15.12 + 1.74 5.88 + 1.41 78.99
MB -> MB + IAA + Kin (1.0) (5.0) Epicot. *66.66 + 7.21 33.33 + 7.21 0
Cot. leaf 16.66 + 8.86 83.33 + 28.86 0

Further, MIK generated plants hardened for roots on MS + 0.1 mg/l NAA (MN) medium, could survive in sterilized soil for 15-20 days. Appearance of one to two new leaves was noticed in regenerated plants in soil and the plants were found to be sensitive to CGMMV.

Discussion. Pusa Madhuras, a cultivated variety of Cucumis melo L. has been found to be responsive to in vitro regeneration. Similar studies have also been reported in other species and varieties of Cucumis melo (4, 5, 6, 7, 8, 9). Somatic embryogenesis has also been noticed in ‘Pyonet Piel de Sapo’, ‘Amarillo canario’ and “Amarillo Golda’ of Cucumis melo (10, 11) and in Pusa Madhuras (12).

Regenerated plants resistant to CGMV and isolated as somaclonal variant would be of immense use in future disease resistance programs.

Literature Cited

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