www.thaiagj.org Thai Journal of Agricultural Science 2007, 40(3-4): 159-166

Inheritance and Ultrastructure of Variegated Leaf Mutant in Mungbean (Vigna radiata (L.) Wilczek)

C. Sangsiri1, W. Sorajjapinun2 and P. Srinives3*

1Center for Agricultural Biotechnology, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand

2Asian Regional Center AVRDC - the World Vegetable Center, Kasetsart University Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand

3Department of Agronomy, Faculty of Agriculture at Kamphaeng Saen Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand

*Corresponding author. Email: agrpss@yahoo.com

Abstract

A variegated leaf mutant of mungbean (Vigna radiata) was derived from gamma irradiation of F1 seed from the cross KPS 2 x VC 6468-11-1B. The mutant was purified until M8 and crossed with the male parent VC 6468-11-1B for inheritance study. All F1 plants revealed normal green leaves while the F2 plants segregated well in a 3:1 ratio of green to variegated plants. The number of F3 lines showing all green plants: segregating plants: variegated plants fitted well with a 1:2:1 ratio. Therefore, it can be concluded that the variegated leaf character was controlled by a single recessive nuclear-encoded gene. Although, the Chl a/b ratio of the mutant was approximately the same as those of the parents, its average chlorophyll a (Chl a), chlorophyll b (Chl b) and total chlorophyll (TChl) contents in the leave were significantly lower. Thick sections of the mutant showed less number of chloroplasts containing in the structure, as compared to the parents. In the mutant tissue, fewer chloroplasts were found in the upper palisade cell layer while more of them were in the lower spongy mesophyll. The ultrathin section of the green sector of the mutant leaf was the same as those of the normal leaf in the character of double chloroplast membrane, stacked grana, thylakoid lamellae, thylakoid system, vacuole, starch grain and osmophilic granule (plastoglobuli). While the ultrastructure of the defective chloroplast in the yellow sector showed large vacuoles with disrupted grana and lamellar systems. There were no starch grain and osmophillic granule. This information can be used in biochemical and photosynthetic study in mungbean in the future. Keywords: Vigna radiata, mungbean, mutant, variegated leaf, ultrastructure

Introduction

Mungbean (Vigna radiata (L.) Wilczek) (2n=2x=22)

is a self-pollinated legume originated in South Asia. It is an economically important crop in China, India, Myanmar, Pakistan, Thailand, and Vietnam, with the combined planted area of over 5 million ha. The crop is considered rather wild and still gives

low seed yield (<1 t ha-1), with uneven maturity. This opens an ample room for mungbean breeders to improve the crop. Besides natural genetic variation available in mungbean germplasm collections, mutation techniques are proven useful in obtaining novel traits and creating genetic variability. Gamma irradiation as a mutagen can induce useful as well as harmful mutation in plants

160 C. Sangsiri et al. Thai Journal of Agricultural Science

(Gupta, 1996; Micke and Donini, 1993). Singh and Sharma (1993) isolated a few pentafoliate and tetrafoliate mutants from the gamma rays- and EMS-treated mungbeans. These mutants showed a significant increase in dry matter production, total chlorophyll content and yield in M2 and M3 generations, as compared to their parents. Characterization of the mutant plants is a powerful approach to understanding the genetic control of plant growth and development. Variegated mutations have been found and studied in many crops because of their relationship with photo- synthesis and chloroplast development. Genetically controlled patterns of somatic variegation were reported in leaves, flowers, and seeds of many crops as the result of unstable alleles in nuclear genes. Santos (1969) and Bahl and Gupta (1984) described the mutant characters and their inheritance in mungbean and reported that albino, chlorina, multifoliata, unifoliata, variegated, and xantha were each controlled by a recessive gene. However, there is only a little knowledge on genetic control of pigment mutants and their ultrastructure in mungbeans.

A variegated leaf mutant C65-6P in mungbean was obtained by exposure of F1 seed of the cross between KPS 2 and VC 6468-11-1B to gamma radiation (Sangsiri et al., 2005). The mutant was allowed to self-pollinate for 7 consecutive generations until M8. The objectives of this study are (1) to determine mode of inheritance of the mutant, and (2) to determine ultrastructure of the mutant leaf sections as compared to those of the normal green leaf

Materials and Methods Study on Inheritance of the Variegated Leaf Mutant

Crosses were made between the mutant and the parent VC 6468-11-1B, a large-seeded high-yielding mungbean line. The F1 seeds were sown in the field and the F1 plants were individually harvested at maturity. They were sown in families of F2 and number of plants with normal green leaf and variegated leaf were recorded at early flowering stage. In one segregating family, individual F2 plants were harvested and sown as plant–to–row to observe for number of F3 plants segregating for normal and variegated leaves in each family. The

observed number was tested against the expected number by using the Chi-square goodness-of-fit test (Mather, 1951).

Field cultural practices in this experiment were conducted based on the standard management for mungbean grown in Thailand. Briefly, the seeds were sown in rows of 50 cm apart using one seed per hill, 12.5 cm between hills of the same row. Weeds were controlled by pre-emergence spraying of imazathapyr at 250 g(ai) ha-1. Late weeds were eradicated by hand-weeding twice at 15 and 30 days after sowing. Insects were controlled by spraying with triazophose (Hostathion 40% EC) at the rate of 40 cc per 20 liters of water when the insect population was building up beyond the threshold level. Irrigation water was applied during the cropping season as needed.

Study on Chlorophyll Content

Chlorophyll content was determined from leaves of each plant at the 4 node stage. Ten plants each were taken from KPS 2 and VC6468-11-1B, while 30 plants were randomly taken from the variegated ones. Each leaf was outlined on a piece of standard paper, cut the paper according to leaf-shape, weighed the paper, calculated the leaf area, then established the relationship between leaf area and paper weight.

Put 3 leaflet in 40 mL of DMF (N, N-Dimethy- formamide) in a glass tube, closed the tube with aluminum foil, then stored in the dark at 4oC for 24 h. The chlorophyll solution was checked for its absorbance by a spectrophotometer at the wave length of 647 and 664 nm. The data were used to calculate the chlorophyll content according to the formula of Moran (1982):

Vol100) Area X(

)12.64A (-2.99A 664647 Chl ×××

+=a

Vol100) Area X

)5.60A (23.26A (

664647 Chl ×

××−

=b

Vol100) Area X(

)7.04A (20.27A 664647 TChl ×××

+=

Where Chl a = Chlorophyll a concentration (g m-2)

Chl b = Chlorophyll b concentration (g m-2) TChl = Total chlorophyll concentration (g m-2)

Vol. 40, No.3-4, 2007 Inheritance and ultrastructure of leaf mutant in mungbean 161

A647 = Absorbance at 647 nm A664 = Absorbance at 664 nm Vol = Volume of DMF (mL) X = Dilution ratio Area = Area of the extracted leaves (10-4 m2)

Study on Comparative Ultrastructure of the Variegated Leaf Mutant

Thick sections from normal leaves of KPS2 and VC6468-11-1B and from the variegated mutants were examined. The mutant leaves were sectioned in both green and yellow tissues. Each leaf was cut to 2-3µm thick by zinc knife, mounted on glass slides and stained by iodine solution. The samples were observed under an Olympus BX 41 phase contrast microscope and photographed.

The protocol for ultrastructure study followed Bonnema et al. (1995). The leaf tissues were observed from 1 × 1 mm2 leaf pieces fixed overnight in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7. Each piece was washed in 0.1 M phosphate buffer, pH 7 and post fixed in 2% OsO4, dehydrated in ethanol, infiltrated with Spurr’s low-viscosity resin for five days, and embedded flat in aluminum weighing dish. Ultrathin sections were mounted on 300-mesh uncoated grids and stained with uranyl acetate and lead solution. Specimens were examined under a JOEL JEM-1230 (Joel Ltd.) transmission electron microscope.

Results

Inheritance of the Variegated Leaf Mutant

All F1 plants showed normal green leaves without reciprocal effect while the F2 plants segregated well in a 3:1 ratio (Table 1). The variegated plants were rather weak and a few of them died at the seedling stage. This caused the proportion of variegated plants in each F2 family to be slightly less than the theoretical ¼, and finally caused significant deviation from 3:1 in the pooled data of the direct cross, although the segregation number were homogeneous among families (Table 2). The number of F3 lines showing all green plants: segregating: all variegated plants fitted well with the 1:2:1 ratio (Table 3). Thus it can be inferred that the variegated leaf character is controlled by a single recessive nuclear-encoded gene. We proposed var1 as the gene symbol.

Table 1 Chi-square values for goodness-of-fit to a 3:1 ratio of the F2 progenies from the cross between normal green leaf mungbean VC 6468-11-1B and variegated leaf mutant C65-6P, and their reciprocals.

Number of F2 plants

FamiliesNormal

leaf Variegated

leaf

χ 2 (3:1) Prob.

Variegated leaf × Normal leaf (direct cross) 1 103 23 3.06 0.08 2 130 37 0.72 0.40 3 138 35 2.10 0.15 4 94 25 1.01 0.32 5 120 33 0.96 0.33 6 83 19 2.21 0.14 7 95 28 0.33 0.57 8 80 24 0.20 0.65 9 99 34 0.02 0.89

10 71 22 0.09 0.77 11 120 37 0.17 0.68 12 115 29 1.81 0.18 13 93 23 1.65 0.20 14 130 37 0.72 0.40 15 122 30 2.24 0.13

Total 1593 436 13.34 0.003 Normal leaf × Variegated leaf (reciprocal cross)

1 84 19 2.36 0.12 2 65 26 0.62 0.43 3 103 31 0.25 0.62

Total 252 76 3.23 0.44

Chlorophyll Content The average Chl a, Chl b and TChl content of

variegated leaf mutant line were 0.17±0.07, 0.07±0.04, 0.24±0.10 g m-2, while those in the normal leaves from KPS2 and VC6468-1-1B were 0.41±0.04, 0.14±0.01, 0.56±0.05 and 0.43±0.05, 0.15±0.02, 0.58±0.07, respectively. However, the ratio of Chl a/b in the mutant and parents remained the same at approximately 2.77-2.91 (Table 4).

162 C. Sangsiri et al. Thai Journal of Agricultural Science

Table 2 Test for homogeneity of χ2 among F2 families from the cross between normal green leaf mungbean VC 6468-11-1B and variegated leaf mutant C65-6P.

Sources of variation

df χ2 Chi-square values at P0.05

Direct cross

Deviation 1 13.34** 3.84

Heterogeneity 14 3.97 25.00

Total 15 17.31 23.68

Reciprocal cross

Deviation 1 0.59 3.84

Heterogeneity 2 2.64 5.99

Total 3 3.23 7.81

Comparative Ultrastructure of the Variegated Leaf Mutant

The variegated plants showed different sectoring patterns, suggested a random expression of the mutant gene at all stages of leaf development. Expression of the trait is highly dependent on light intensity. The leaves developed under shaded canopy showed more green sectors than the yellow ones and vice versa for those developed under higher light intensity canopy (Figure 1). The thick sections of KPS 2 and VC 6468-11-1B normal mungbean showed more chloroplasts than the variegated mutant in the same size of microscopic film. In the mutant tissue, less chloroplast was found in the upper palisade cell layers while more of them were in the lower spongy mesophyll. The part that expressed yellow color was the two upper most palisade cell layers (Figure 2).

Table 3 χ2 values for goodness-of-fit to a 1:2:1 ratio of one representative F3 lines from the cross between variegated leaf and normal leaf mungbeans.

Leaf traits Observed (O)

Expected (E)

χ2

(O-E)2/E P-value

Normal leaf lines 38 39.25 0.040

Segregated F2 lines 78 78.5 0.003

Variegated lines 41 39.25 0.080

Total 157 157 0.123 0.94

Table 4 Chlorophyll content of normal green leaf mungbean lines, KPS 2 and VC 6468-11-1B, compared with the variegated mutant line, C65-6P1/.

Chlorophyll a(Chl a)

Chlorophyll b(Chl b)

Total Chlorophyll Mungbean genotypes

(----------------------- g m-2--------------------------)

Chl a: Chl b ratio

KPS 2 0.41 a 0.14 a 0.55 a 2.91 a

VC 6468-11-1B 0.43 a 0.16 a 0.58 a 2.77 a

Variegated (C65-6P) 0.17 b 0.07 b 0.24 b 2.88 a 1/ Values followed by the same letter within a column are not significantly different from each other

according to DMRT at the 5 % level of probability.

Vol. 40, No.3-4, 2007 Inheritance and ultrastructure of leaf mutant in mungbean 163

a b

c d

Figure 1 A variegated leaf mutant C65-6P showed various expressions of green and yellow sectors when the leaves were exposed to high (a), medium (b), and low (c) light intensity in the canopy, (d) compared with its progenitors, KPS 2 and VC 6468-11-1B.

a b

c d

e f

Figure 2 Somatic mutation in leaves of C65-6P. (a) Micrograph of a cross-section from mutant leaf sector without staining, (b) stained with iodine solution. The upper palisade cell layer (UPa) is yellow while the rest of cell layer is green. (c) Micrograph of a cross-section from normal green KPS2 leaf without staining, (d) stained with iodine solution. (e) Micrograph of a cross-section from normal green VC6468-11-1B leaf without staining, (f) stained with iodine solution.

164 C. Sangsiri et al. Thai Journal of Agricultural Science

The ultrathin section of a normal leaf (Figure 3) showed double chloroplast membrane, stacked grana, thylakoid lamellae, thylakiod system, vacuole, starch grain and osmophilic granule (plastoglobuli), which were responsible for electron transport during photo- synthesis. Ultrastructure of the green sector of the mutant leaf was the same as that of the normal one. However, the yellow sector of the mutant leaf showed larger vacuoles with disrupted grana and lamellar systems. There was neither starch grain nor osmophilic granule found in the tissue, while different plastids in the mutant leaf were of the same size (approximately 3 µm in diameter) as in the normal one.

Discussion

Variegation is a frequently observed mutant in

plants that can be controlled by either nuclear or cytoplasmic genetic alteration. The inheritance of variegated leaf is significant in increasing knowledge related to photosynthesis. There were a number of

reports on genetic analysis of variegated mutants in alfalfa (Smith, 1989), Arabidopsis (Martínez-Zapater, 1993; Rodermel, 2002; Sakamoto, 2003), carrot (Miller et al., 1984), maize (Walbot and Coe, 1979), mungbean (Bahl and Gupta,1984; Santos, 1969), pea (Miller et al., 1984), soybean (Cheng and Chandlee, 1999; Honeycutt et al.,1990; Nissly et al., 1981; Palmer et al., 1990, 2000), and tree tobacco (Marcotrigiana and Hackett, 2000). It can be concluded that there are many loci of the variegated mutant genes in different plant species, some with multiple alleles. For example, var2 mutant locus in Arabidopsis has 10 alleles (Rodermel, 2002). So far, there were 2 previous publications on chlorophyll mutation in mungbean which were reportedly controlled by nuclear recessive-single gene (Bahl and Gupta, 1984; Santos, 1969). Although our variegated leaf mutant is also conditioned by a nuclear recessive gene, we have no clue whether it is the same mutant as previously reported. The previous reports did not study on ultrastructure of the mutant leaves and

a b

c d

Figure 3 Electron micrograph of chloroplasts. (a) A chloroplast from normal green leaf KPS 2 exhibited a thylakoid system with well-developed grana (G), thylakoid lamellae (L), osmiophilic granules (OG) and starch grains (S) (bar = 1µm). (b) A chloroplast from normal green leaf VC6468-11-1B exhibited similar features to those of KPS 2 leaf (bar = 1µm). (c) A green sector from mutant leaf exhibited the same features as normal green leaf (bar = 0.5µm). (d) A chloroplast from yellow tissue exhibited large vacuole (V), unstacked grana, unstacked thylakoid membrane structure, no OG and no starch grain (bar = 0.5µm).

Vol. 40, No.3-4, 2007 Inheritance and ultrastructure of leaf mutant in mungbean 165

thus we have no detail about disruption of the organelles which are responsible for photosynthesis.

The average Chl a, Chl b, and TChl of variegated leaf was significantly less than those of the normal leaf, however the Chl a/b ratio was the same in both leaf types. There were some mutant plants showing higher Chl a/b ratio than the normal ones. This revealed that rate of change in the content of Chl a and Chl b was not the same among the mutants, possibly was due to the impair of Chl b synthesis during chloroplast development. Increases in Chl a/b ratio in mutant plants were reported earlier by Darr and Arntzen (1986), Picaud and Dubertret (1986) and Tanaka et al. (1993). Chlorophyll synthesis in the mutant seemed to be environmentally dependent, especially on light intensity during the growing period. Under less light intensity, the mutant produced more chlorophyll and thus became greener (Figure 1). The structure of mutant leaf is normal, consisting of upper palisade cell layers, lower spongy mesophyll and lower epidermis. However, fewer chloroplasts were found in the upper palisade cell layers while most of them were in the lower spongy mesophyll. The two upper most palisade cell layers are responsible for the yellow variegation, similar to what previously described in v2 mutant in soybean (Cheng and Chandlee, 1999).

The ultrastructure of variegated leaf showed no thylakoid system with many vacuoles in each cell. The disrupted chloroplasts looked like am mutant in Arabidopsis (Rodermel, 2002) and Chl mutant in tree tobacco (Marcotrigiano and Hackett, 2000). Without the thylakoid system photosynthesis in the yellow and white tissue was totally impaired, and neither starch nor plastoglobuli was produced. Thus this mutant is useful as a material for further study in terms of organelle function and biochemical process related to plant photosynthesis.

Acknowledgments

This study is a part of Ph.D. thesis of the senior

author who is granted the Royal Golden Jubilee Ph.D. Scholarship from the Thailand Research Found. Parts of the experiment were supported by Center for Agricultural Biotechnology, Kasetsart University; the Thailand Research Fund; and Thailand’s National Center for Genetic Engineering and Biotechnology.

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Manuscript received 21 November 2007, accepted 26 April 2008

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