Selection of Flower Buds and Carbon Source for Anther Culture in Melon (Cucumis melo)

A K M A Islam*, Shuji Misoo** and Takeshige Ishii***

Pollen fertility studies showed that flowers from the early flowering stage contain more fertile pollen (93.9-97.6 %) than later stage of flowering (68.9%). Pollen fertility percentage indicates development of anther. If the anthers are well-developed, the pollen fertility is expected to be high. The uninucleate stage is optimal for the culture of melon anthers, and these pollen stages appear in flower bud and anther with 4.5-5.0 mm and 1.9-2.0 mm length, respectively, in first week and 3.3-4.3 mm and 1.6-1.8 mm length, respectively, in the second week. This result indicates that flower and anther size reduces with increase in the age of the donor plant. In the early flowering stage, the plant produces larger flowers than at later stages. Among the three polysaccharides, lactose showed good effect on pollen division. The anther cultured in lactose supplemented MS medium produced the highest number of living anther per plate, number of living pollen per anther and dividing pollen per anther. Maltose supplemented MS medium showed moderate performance and sucrose the lowest for all parameters studied.

Keywords: Melon (Cucumis melo L.), Bud selection, Pollen fertility, Uninucleate stage, Anther culture

Introduction The most common name used for Cucumis melo L. is melon. Other names include sweet melon, round melon, muskmelon, casaba, cantaloupe and winter melon (Robinson and Decker-Walters, 1997; and Nayar and Singh, 1998). Melon is a member of genus cucumis under the family cucurbitaceae (Mallick and Masui, 1986). It has many uses, such as immature melon used in fresh salad, cooked (soup, curry, stew and stir-fry) and pickle. Mature fruits are eaten fresh as a dessert fruit, canned or used for jam. Melon has a basic chromosome number of 12 and is a diploid species, 2n=24 (Kroon et al., 1979). Spontaneous haploid in melon occurs rarely. Haploid by anther culture can be used for the improvement of melon. It needs to give attention on the improvement of melon developing 1) high quality flesh with disease and insect resistance; 2) shifting to monoecious and

gynoecious types for F1 hybrid seed production; 3) novel fruit types to broaden the utilization of melon; 4) novel plant type for home garden; 5) concentrated fruit set once- over harvest and 6) earliness. The culture of anther in vitro or on an artificial growth media is called anther culture. First successful anther culture was reported by Guha and Maheshwari (1964 and 1966) in

* Associate Professor, Department of Genetics and Plant Breeding, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur 1706, Bangladesh. E-mail: xxxxxxxxxxxxxxxxxxx ** Faculty of Agriculture, Laboratory of Plant Breeding, Kobe University, Japan. E-mail: xxxxxxxxxxxxxxxxxxx Author plz provide *** Faculty of Agriculture, Laboratory of Plant Breeding, Kobe University, Japan. E-mail: xxxxxxxxxxxxxxxxxxx mail ids

©Selection 2011 IUP. of AllFlower Rights Buds Reserved. and Carbon Source for Anther Culture in Melon (Cucumis melo) 1 Datura innoxia, and has been modified by various workers (Sopory and Munshi, 1996). Nowadays anther culture technique is extensively used in haploid breeding of many economic crops like rice, wheat, maize, barley, potato, Brassica, asparagus, etc. This is a relatively simple and efficient technique for haploid production. Haploid plants have the gametic number of chromosomes. Haploid plants are of special interest to plant breeders because of the possibility of utilizing their chromosome doubled derivatives in breeding programs as inbred lines. The success of anther culture depends on the size of flower buds and the developmental stage (mid-uninucleate to late uninucleate) of pollen grain. At uninucleate stage, microspores are most suitable for the induction of androgenesis. So the present investigation was undertaken to standardize the flower bud size and developmental stage of pollen grain of melon for successful anther culture.

Materials and Methods Establishment of mother plant: The melon plant was cultivated in a glass house under favorable temperature, light, relative humidity and photoperiod (Figure 1a). Selection of material for anther culture: The closed flower buds were excised for the study from the top of the plant because anthers of flower buds from other parts of the plant are usually not good for culture. Assessment of pollen fertility: The anther of excised flower buds were placed on a drop of acetocarmine on the slide, broken and kept lying 1-2 min. Afterwards the preparation was squashed by means of a coverslip, the slide was observed under the microscope and the number of normal (stained) and abnormal (non-stained) pollen was recorded. Assessment of anther development stage: Harvested flower buds of different length with different anther length were used to prepare a slide in acetocarmine. The slides were observed under microscope to find out mid-uninucleate to late uninucleate stage. About 200 flower buds were studied. Preservation of flower bud: Flower buds of 3.5-5.0 mm in length with 1.6-2.0 mm anther length were picked, wrapped in beaker in a small amount of water and preserved in refrigerator at 6 °C for 3-4 days. Sterilization of flower bud: The flower buds were dipped in 70% ethanol in a beaker for 30 s. The alcohol was discarded and 2% NaOCl with one drop of Tween 20 were added. The flower buds were left in the beaker for 8 min; the beaker was shaken and then rinsed twice in pure distilled water. Inoculation of anther and incubation of culture: The anthers were stripped, the filament remnants were discarded and immediately placed on the culture medium. Among the three anthers, only two big anthers were placed on the culture medium by touching the inner side on the medium. About five anthers were inoculated in each petri dish. The

2 The IUP Journal of Genetics & Evolution, Vol. IV, No. 3, 2011 anthers should be well distributed on the medium. The inoculated anthers were kept in the incubator at 30 °C day temperature, 25 °C night temperature, 14 h light and 10 h dark. Assessment of pollen viability and pollen division: The anthers were inoculated in modified MS media supplemented with lactose, maltose and sucrose. Ten days to two weeks after inoculation, 15 anthers were taken from three plates and slide were prepared. The slides were observed under microscope and the number of viable (stained) and dividing pollen grain in each preparation was counted.

Results and Discussion The detailed results of this experiment are presented in Tables 1-3. Data were not subjected to statistical analysis, only the mean values are given in tables.

Assessment of Pollen Fertility The pollen fertility results in each of the four weeks are presented in Table 1. Three flowers were studied in each week. The results showed that flowers from the early flowering stage contain more fertile pollen (93.9-97.6%) than later stage of flowering (68.9%). Pollen fertility percentage indicates the development of anther. They are more or less correlated. If the anthers develop well, the pollen fertility is expected to be high. Correlation between optimal development of anther and pollen fertility percentage is absolute.

Table 1: Assessment of Pollen Fertility at Different Growth Stages in Melon Total No. Flower No. No. of Total No. No. of Total % of Weeks from Each Normal of Normal Abnormal No. of Pollen Abnormal Sample Pollen Pollen Pollen Pollen Fertility Pollen 1 129 5 1 2 37 199 6 13 212 93.9 3 33 2 1 43 1 2 2 53 163 2 4 167 97.6 3 67 1 1 39 2 3 2 42 111 4 8 119 93.3 3 30 2 1 28 13 4 2 23 82 8 37 119 68.9 3 31 16

Selection of Flower Buds and Carbon Source for Anther Culture in Melon (Cucumis melo) 3 Assessment of Pollen Developmental Stage The uninucleate stage is optimal for the culture of melon anthers. Nitsch and Nitsch (1969) reported that only anther which contained pollen at uninucleate stage shows better response in anther culture. In this study, uninucleate, binucleate and tetrad pollen stage (Figure 1b, 1c and 1d, respectively) appeared in flower bud. Figure 1: Melon Plant with Flower, Pollen Developmental Stages and Pollen Division in Anther Culture

Melon Plants with Flower Uninucleate Stage Binucleate Stage

Tetrad Stage Multinucleate Stage Multinucleate Stage The pollen developmental stage varied with the age of the flowering plants. In the first week, suitable pollen developmental stage for anther culture was observed in flower bud length 4.5-5.0 mm with 1.9-2.0 mm anther length. But in the second week, suitable pollen stage was observed in flower bud length of 3.3-4.0 mm with anther length of 1.6- 1.8 mm (Table 2). The results indicate that flower and anther size reduces with increase in the age of the donor plant. In the early flowering stage, the plant produces larger flowers than at later stages. So, the pollen developmental stage needs to be checked at every Author pl chk not mentioned time of anther culture regarding the age of donor plant as well as flowering stage. Guha in the ref. list and Mukharjee (1973), working with 20 rice cultivars, pointed out that the young rice anthers at the tetrad or early stage failed to develop and that anther containing only uninucleate microspores showed differentiation into pollen embryos. The uninucleate stage was found to be optimal for a number of species (Maheshwari et al., 1980 and 1982). Assessment of Pollen Viability and Pollen Division Pollen viability and pollen division upon culture were enhanced by the preculture treatment

4 The IUP Journal of Genetics & Evolution, Vol. IV, No. 3, 2011 Table 2: Assessment of Pollen Developmental Stage at Different Growth Stages in Melon

First Week Second Week Bud Petal Anther Dev. Petal Anther Dev. Stage Anther Anther No. Length Length Stage of Length Length of Pollen No. No. (mm) (mm) Pollen (mm) (mm) 1 5.0 1 1.9 BN 4.5 1 1.9 BN

2 1.9 BN 2 1.9 BN

2 3.5 1 1.2 T 4.8 1 2.0 BN

2 1.1 T 2 2.0 BN

3 4.0 1 1.6 EUN 4.3 1 2.0 BN

2 1.5 EUN 2 2.0 BN

4 4.2 1 1.9 EUN 3.2 1 1.7 EUN

2 1.7 EUN 2 1.8 EUN

5 4.5 1 2.0 EMUN 3.4 1 1.6 LUN

2 2.0 EMUN 2 1.7 LUN

6 4.7 1 2.0 LUN 3.0 1 1.8 MUN

2 2.0 LUN 2 1.8 MUN

7 4.5 1 1.9 EUN 3.2 1 1.6 MUN

2 1.9 EUN 2 1.7 LUN

8 4.0 1 2.0 MUN 5.0 1 2.1 BN

2 2.0 MUN 2 2.1 BN

9 4.3 1 2.0 LUN 4.8 1 2.0 BN

2 1.9 MUN 2 2.1 BN

10 4.0 1 1.9 LUN 4.6 1 2.1 BN

2 1.9 LUN 2 2.2 BN

Note: BN=Binucleate; T=Tetrad; EUN=Early Uninucleate; MUN=Mid-Uninucleate; LUN=Late Uninucleate; and EMUN=Early to Mid-Uninucleate.

(cold treatment) and carbon sources of culture media. Table 3 reveals that among the two pretreatment, flower buds preserved at 6 °C for 3-4 days prior to anther culture showed

Selection of Flower Buds and Carbon Source for Anther Culture in Melon (Cucumis melo) 5 Table 3: Assessment of Pollen Viability and Pollen Division in Anther Culture in MS Media Supplemented with Three Different Polysaccharides After Two Weeks No. of No. of No. of No. of No. of No. of Non- Total No. Culture Anthers Anthers Living Dividing Treatment Anthers Dividing of Pollen Media Culture Live/ Pollen/ Pollen/ Callusing Pollen/ /Slide /Plate Plate Anther Anther Anther Lactose 5 3a 0 55.6a 18.6a 37.0a Without Preservation Maltose 5 2b 0 4.0b 0.20b 3.8b Sucrose 5 0c 0 0.0c 0.0c 0.0c 3,455.7 Lactose 5 4a 0 128.0a 25.6a 102.4a With Preservation Maltose 5 1b 0 16.0b 1.8b 14.2b Sucrose 5 1b 0 0.4c 0.4c 0.0c good effect on pollen division. On the other hand, lactose showed improved pollen division in anther culture (Table 3), compared to the response of sucrose and maltose. The highest number (4) of living anther was found in the anther culture in MS medium supplemented with lactose after four days’ preservation of flower bud. The highest number of living pollen per anther (128) and dividing pollen per anther (25.6) was also found in the lactose supplemented treatment. Maltose showed moderate performance and sucrose showed the lowest for all the parameters studied. The total number of pollen grain was counted on an average from one preparation and it was 3,455.7. Dividing pollen contains different number of cells such as 2c, 3c, 4c, 5c, 6c, 7c, 11c and 15c (Figure 1). ^

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