Micropropagation of Watermelon

by:  Tarek Yehia Kapiel
[Reproduced here with permission from Tarek Kapiel; see PowerPoint presentation at end of text]

Rapid Micropropagation of Diploid and Tetraploid
Watermelon (Citrullus lanatus) Cultivars

Mahmoud I. Nasr¹, Ibrahim A. Ibrahim¹, Hala M. Habib² and Tarek Y. Kapiel²
1Genetic Engineering and Biotechnology Research Institute (GEBRI), Sadat City, Minufiya University, Egypt.
2. Botany Department, Faculty of Science, Cairo University, Egypt.

Abstract

A simple and rapid protocol for micropropagation of diploid and tetraploid watermelon (Citrullus lanatus) using shoot-tip and nodal cuttings explants was achieved. Shoot tips explants from 6-14 days aseptically germinated seedlings were cultured on solidified MS media containing test concentration of benzyladenine BA at (0.0, 0.25, 0.50, 1.0, 2.5 & 5.0 mg/l) and indole-3-butyric acid (IBA) at (0.0, 0.1, 0.2, 0.3, 0.4 & 0.5 mg/l), for 3 weeks. MS medium supplemented with 1.0 mg/l BA showed the highest percentage of shoot formation. MS medium supplemented with 0.5 mg/l IBA was the best medium for both shoot and root proliferation. The well established protocol involved the stimulation of axillary-bud development from excised seedling shoot tips by a high cytokinin (BA) on MS medium, depending on genotype and ploidy level, with subculture periods of 18 to 21 days, then directly root the shoots buds, with or without a shoot elongation step on 0.5 mg/l I?BA for 12 to 21 days. Well-rooted plantlets were successfully transplanted to 1:1 peat:sand substrate and acclimatized in the lab under cool white light under a clear plastic film tent for 7-10 days and gradually hardened-off to green house conditions, after which they could be transferred to field conditions. The effect of various factors on plant regeneration of watermelon was also studied to enhance the established protocol.

Introduction

In vitro procedures are used to some degree in almost every major agronomic, vegetable and fiber species. The success of such technology requires an efficient protocol for plant regeneration from isolated organs, tissues, and cells. Watermelon, Citrullus lanatus, is an important food crop that has received less attention in this area than the closely related cucumber, C. sativus. However, the literature descriptions of micropropagation methods applied to Watermelon, C. lanatus, are few.

Tissue culture represents a promising tool for use in C. lanatus breeding schemes. Recent cell and tissue culture research has resulted in an efficient plant regeneration system for watermelon (Compton and Gray, 1993; Dong and Jia, 1991; Srivastava et al., 1989). The published protocols use seedling cotyledons as a source of explants, from which adventitious shoots can be obtained by the process of organogenesis. The shoots can be rooted to form complete plants in 6-10 weeks from culture initiation. These protocols have been used both to obtain model transgenic plants and to obtain tetraploid plants, which can be used to breed improved triploid watermelon cultivars (Compton and Gray, 1994). Techniques to micropropagate triploid and tetraploid watermelon from shoot tips and meristems have been known for many years (Xu et al. 1979; Anghel and Rosu 1985). Mutations or off-types have not been reported from culture of these predetermined buds. Barnes 1979 developed a 3-stage, in vitro propagation system for diploid watermelon (cv. Charleston Gray) was also applicable to the economic production of triploid (seedless) watermelon transplants. Helmle-Janosi et al.,1992 Contributed to the micropropagation of triploid watermelon. They found that the size of C. lanatus cv. Erwyn Fl explants affected proliferation. Compton et al., 1993 demonstrated a simple protocol for micropropagating diploid and tetraploid watermelon using shoot-tip explants. The aim of the recent work was to establish a micropropagation and plant regeneration system from different explants (shoot tip and nodal cuttings) of diploid and tetraploid cultivars from local and exotic watermelon.

Materials and Methods

Seeds
Local watermelon cultivars Giza 1 and Giza 21 seeds and two exotic watermelon cultivars (Odem, Aswan) seeds were used in this investigation. Although, another 20 different genotypes (13 tetraploid and 7 diploid) which are kindly provided by Prof. Dr. Bill Rhodes, Clemson University, were evaluated under optimum conditions of our established protocol.

Establishment of aseptic culture
The tissue culture work was conducted at Genetic Engineering and Biotechnology Research Institute (GEBRI) at Sadat city, Minufiya University during the period from June 1998 to December 1998. The work included the following points:

Surface sterilization
Watermelon seeds were surface sterilized by using the best method obtained for surface sterilization (unpublished data). Seeds were dipped for 20 minutes in HCl at 20% and NaOCl at 1% for 20 minutes. All treatments contained five drops of 0.1 % Tween 20. Seeds were then rinsed three times using sterilized distilled water. All steps of sterilization were carried out under aseptic conditions inside the culture cabinet (Laminar Air Flow Hood) and by using sterilized instruments. Watermelon seeds were cultured on Murashige and Skoog (MS) basal medium supplemented with 10 g/l sucrose, 0.1 mg/l thiamine HCl, 0.1 mg/l nicotinic acid 0-5 mg/l pyridoxin and 100 mg/l myo-inositol. Seeds were cultured individually in test tubes, and 25 seeds were used to determine the germination percentage of each genotype. All cultures were maintained in growth chamber at 30 + 2 ° C.

After germination the shoot tip explants and nodal cuttings of all tested seeds were used for micropropagation studies. Shoot tip explants from 6-14-day-old aseptically germinated seedlings were cultured on solidified MS medium containing test concentrations of BA or IBA or both as follows:

This experiment was conducted to study the effect of BA and IBA on plant growth and development from shoot tip explants from 6-14-day-old aseptically germinated seedlings of cv. Giza 21. Explants were cultured on solidified MS medium containing test concentrations of benzyladenine (BA) at (0.0, 0.25, 0.50, 1.0 2.5, and 5 mg/l), or indole butyric acid (IBA) (at 0.0, 0.1, 0.2, 0.3, 0.4 and 0.5 mg/l). All cultures were maintained in growth chamber at 30 + 2 ° C for 3 weeks in a 250-ml jars. Five explants were cultured in each jar and 10 replicates for each genotype were used. Data of the experiment were analyzed using PC-Stat (1985) package, and Treatment effect and The Least Significant Difference (LSD) test were calculated by using One Way Analysis of Variance (Anova) General test.

2.  Effect of different concentrations and combinations of both BA and IBA

This experiment was conducted to study the effect of different concentrations and combinations of both BA and IBA on plant growth and development from shoot tip explants from 6-14-day-old aseptically germinated seedlings of cv. Giza 21. Explants were cultured on solidified MS medium containing test concentrations of benzyladenine (BA) at (0.0, 0.25, 0.50, 1.0 2.5, and 5 mg/l), or indole butyric acid (IBA) (at 0.0, 0.1, 0.3, and 0.5 mg/l). The same procedures and statistical analysis were applied as in the above experiment.

II. The Effect of various factors on plant regeneration of watermelon (Citrullus lanatus).

After establishment of aseptic cultures, the effect of genotype (Giza 1, Giza 21, Aswan, Odem), explant type (Shoot apex, single node with one leaf and single node), explant age (14, 21 and 28 days), on plant regeneration (shoot elongation, average number of leaves and rooting) were studied. On the other hand, the effect of some components of plant tissue culture nutrient medium such as MS-salt strength (25, 50, 75 and 100%), sucrose (1, 2 and 3%) and gelling agent (8 g/l agar, 2 g/l gelrite and 4 g/l agar + 1 g/l agar + 1g/l gelrite) on plant regeneration (shoot elongation, average number of leaves and rooting) were also studied. The same procedures for experimental design and statistical analysis were applied as previously described.

Results
Establishment of aseptic culture:

The objective of this stage is simply to attain all aseptic tissue culture of watermelon. It is only necessary that the culture be free from obvious infection that a suitable proportion of explants survives in culture conditions, and that there is rapid growth among the explants. The following data (Table 1) were recorded after 2 weeks from culturing. Data were reported in terms of percentage of germination and the most pronounced response for each genotype. It was found that, germination percentage depends upon the genotype and the ploidy level of the tested seeds. In general, diploid seeds have higher percentage of germination than the tetraploid ones. However, the highest germination percentage was for the diploid, local cultivars Giza 21 thus it has been selected as a starting explants for micropropagation studies.

Micropropagation and In vitro studies

Effect of different concentrations and combinations of plant growth regulators (BA and IBA) on plant regeneration and vitrification from shoot tip explants of Watermelon.

Effect of Benzyl adenine (BA) and Indole butyric acid (IBA).

Table 2 shows the effect of plant growth regulators (BA and I BA ) on plant regeneration and vitrification from shoot tip explants of watermelon (C. lanatus cv. Giza 21). Data obtained showed that, all treatments were highly significant. MS-medium supplemented with 1.0 mg/l BA showed the highest shoot formation and multiplication rates, followed by MS medium supplemented with either 2.5 or 5.0 mg/l BA respectively. On the other hand, as concentration of BA increases the average number of roots decreased and vice versa. Higher concentrations of BA (2.5 and 5.0 mg/l) completely inhibit root formation and induce vitrification. Also, it was observed that higher concentrations of IBA (0.4 and 0.5 mg/l) produced a higher number of shoots per explant with reduction in multiplication rate compared with those obtained in presence of BA. According to the previous results as concentration of IBA increases the average root number increases with significant difference. It should be noticed that IBA does not induce vitrification in watermelon in all tested concentrations.

Effect of different concentrations and combinations of both BA and IBA

Data presented in Table 3 concerning the different combinations between BA and IBA, showed that all treatments are highly significant. The best results of plant regeneration and multiplication were achieved when medium supplemented 5.0 mg/l BA + 0.1 mg/l IBA and MS with 2.5 mg/l BA + 0.1 IBA were used. As a general results the increase of cytokinin generally increase the number of shoots produced as well as vitrification. On the other hand, as the concentration of auxin (IBA) increases the percentages of root formation increases up to 0.5 mg/l. One of the most exciting results is that, MS media supplemented with 0.5 mg/l IBA is the best medium for both shoot and root proliferation with no vitrification, so, it was selected as the best medium for micropropagation and in vitro studies. Table 4 shows the current procedures for watermelon micropropagation. The process involved the stimulation of axillary-bud development from excised seedling shoot tips by a high cytokinin (1.0 mg/l BA) on a MS medium with subculture periods of 18 to 21 days, then to directly root the shoot buds, without a shoot elongation step, on 0.5 mg, IBA for 12 to 21 days depending on genotype and ploidy level. Well-rooted plantlets were successfully transplanted to a 1:1 peat:sand substrate and, acclimatized in the lab under cool white light under a clear, plastic film tent for 7-10 days and gradually hardened-off to greenhouse conditions, after which they transferred to field conditions in Khatatba area and evaluated for both vigor and fruit production. Our successful micropropagation protocol takes from 40-63 days from starting up to transfer to field conditions.

II. The Effect of various factors on plant regeneration of watermelon (Citrullus lanatus).

After establishment of a plant regeneration system for watermelon the effect of various factors on plans regeneration or watermelon (C. Lanatus) were studied to enhance the established protocol. So, many experiments have been designed, and the collecting data are shown in Table 5. From the above mentioned experiments we found that, several factors related to explant selection genotype, age, and media composition have a significant effect and are responsible for great differences in regeneration. Regarding the effect of genotype on growth characteristics (average shoot length, average number of leaves/shoots and average number of roots/shoots) it was found that local cultivar Giza 21 showed the best growth response followed by Giza 1 among tested genotype. Regarding the effect of explant type and age on regeneration characteristics, it was found that shoot apex is the best used explant followed by single node with one leaf explant. Single node with one leaf explant of 21 days old, is superior to the other tested explants. From these results, we were modified stage I of the already established protocol. The protocol may be extends to increase the propagula through nodal cuffing technique, which produce large amounts of plantlets after subculture into rooting media. On the other hand, the effects of three important components of culture media (MS salts, sucrose and gelling agent) were also investigated. Data obtained showed that, half strength MS media is the best-used media compared to all other tested media with no significant difference between quarter or full strength MS-media and 8 g/l agar and 3% sucrose are indispensable.

Discussion

The first reports of regeneration from watermelon cotyledons used both auxin (IAA or NAA) and cytokinins (BA, 2iP or kinetin) to induce regeneration (Barnes 1979; Blackmon and Reynulds 1982; Anghel and Rosu 1985; Wilkins 1985). Cytokinins are often used to stimulate growth and development, kinetin 2ip and BA being in common use. They usually promote cell division especially if added together with an auxin. In higher concentrations, they can induce adventitious shoot formation, but root formation generally inhibited. They promote axillary shoot formation by decreasing apical dominance and they retard aging (Pierik, 1987).

Our results are in accordance with the most recent reports which have shown that benzyladenine was sufficient to induce regeneration, and the auxins 2,4-D, NAA, and IAA promoted excessive callus and reduced shoot formation (Adelberg and Rhodes 1989; Srivastava et al., 1989; Compton and Gray I 993a). Benzyladenine as sole PGR is currently preferred (Adelberg et al., 1997). Results under discussion indicated that addition of BA to MS basal medium improved shoot production from different explants of watermelon. Higher concentrations of BA are liable to induce vitrified roots, which are usually difficult to root. The investigations published so far regarding the effect of auxins and cytokinins on callus productions seemed to be in agreement with these results. Shoot establishment and shoot proliferation can often be satisfactory by cytokinin alone (Read and Young, 1987).

Regarding the effect of IBA on growth characteristics the obtained results showed that IBA at 0.5 mg/l is also preferred to induce root proliferation without remarkable effect on shoot production. It is superior to 1 mg/l BA in that it does not induce vitrification. The results also reveal the stimulating effect of several combinations of cytokinins and auxins, added to the culture medium, on growth characteristics. The obtained protocol (previously described) involved the use of both BA (1 mg/l) at stage I, and IBA (0.5 mg/l) at stage II. The rooted plantlets were successfully acclimatized in green house and evaluated in the field conditions. Genotypic variation in regeneration rates is common (Srivastava et al., 1989; Compton and Gray l993a; Zhang, et al. 1994). Tetraploid and triploid cotyledons generally have lower rates of regeneration (Compton and Gray 1993a). Age of seedling before explant induction varies between laboratories with 3 to 10 days being common. Enhanced branching of apical buds has been described from seedlings (Barnes 1979), immature embryonic axes (Adelberg and Rhodes 1989), and excised meristems (Anghel and Rosu 1985; Gray and Elmstrom 1991). Axillary bud of seedling have also been used (Wilkins 1985; Compton et al., 1993). Nodal cutting explants have not been previously tested as an explant source and they showed the highest frequency of regeneration in the present study. MS medium is very popular, because most plants react to it favorably. However, it should be appreciated that this nutrient solution is not necessarily always optimal for growth and development since the salt content is so high. Shoot formation of watermelon improved well on MS at half strength where the growth parameters recorded the highest values (average shoot length, average number of leaves/shoot and average number of roots/shoot). Data under discussion clearly indicated that MS-medium supplemented with 3% sucrose is the best medium for growth of watermelon plant compared with the other tested media. On the other hand, it is obvious from the obtained data that MS basal medium solidified with 8 g/l agar recorded the best growth of watermelon compared to the other tested concentrations of gelrite and agar + gelrite. The same results were obtained by Singha (1982) working on pyrus communis "Seckel" who noticed that shoot proliferation was significantly greater at higher agar concentrations (1.27%, 0.91% and 0.61%) as compared to 0.3% or lower. The influence of agar is due to its effect on diffusion rate of molecules through the medium. On the other hand, Singha (1982) found that greatest shoot proliferation of Malus sp. "Alem" occurred on medium containing 0.3% agar. Higher agar concentrations decreased shoot proliferation.

References

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