Acta Botanica Sinica 2002, 44 (7):832-837
Graft-induced Inheritable Variation in Mungbean and Its Application in Mungbean Breeding
ZHANG Dan-Hua1, MENG Zhao-Huang2, XIAO Wei-Ming1, WANG Xue-Chen3, SODMERGON1

(1. College of Life Sciences, Peking University, Beijing 100871, China;
2. Shangqiu Research Institute for Agriculture and Forest, Shangqiu 476000, China;
3. College of Biological Sciences, China Agricultural University, Beijing 100094, China)

Received: 2002-03-11 Accepted: 2002-04-26 Supported by a giant from the State Key Basic Research and Development Plan of China (G1999011700) and by the National Science Fund for Distinguished Young Scholars of China (30025004).

Abstract: Graft can induce inheritable variations in the progenies of the scion plants. Seedling of mungbean Vigna radiata (L.) Wilczek) was grafted onto the stem of sweet potato (Ipomoea batatas (L.) Lam.). The growth of the scion was maintained until the scion produced selfed seeds. We sowed the seeds for several generations under normal conditions. Distinct genetic variations appeared in the progenies. Similar variations did not appear in the generations of the scion sowed normally without graft. The variations seemed to be induced by the graft and they inherited steadily. For understanding the possible mechanism of the phenomenon (graft-induced inheritable variation), we analyzed the cytoplasmic and genomic DNA of the variations. The results showed that there was no restriction fragment length polymorphism (RFLP) in the cytoplasmic DNA between the original scion and the variation. However, significant difference between the scion and variation was recognized by random amplified polymorphic DNA (RAPD) analysis. In addition, there was no evidence that indicated the gene transformation from stock to scion. Our results suggest that the non-specific grafting has a pragmatic potential for plant breeding and crop improvement and, the genetic variation seems not to he caused simply by DNA transformation but most likely the stress induced mutation.

Genetic variations emerge in the progenies produced from the selfed fruits of the scion when different species of plant are grafted. Such a phenomenon, called graft-induced genetic variation, has been reported in the 1960's[1-3]. These earlier works use male sterile petunia for stocks and normal fertile petunia for scions. The results show that the male sterility of the stock can be transferred to the progenies of the scion with a certain rate and the male sterility appeared in the progeny of the scion inherits steadily. Later studies in red pepper[4 10], eggplant[11, 12] and soybean[13] give similar results. The common facts shown in these studies are the stock genesis of the variations and that the changed traits seem to behave according to the Mendelian rules in cross-tests. Therefore, it was proposed that the graft-induced genetic variation must be due to gene transformation from the stock to the scion and the reproductive cells of the scion must be the targets of the gene transformation14.

However, current knowledge in cell biology does not support the long distance movement of DNA that is required for the proposition of gene transformation. In the present study, we describe our achievement in mungbean improvement via non-specific graft, suggest the possibility of the technique as a new way for plant breeding and discuss the possible mechanism for graft-induced genetic variation.

1 Materials and Methods

1.1 Graft

Sweet potato (Ipomoea batatas (L.) Lam.) cv. Zhenghong 9 (ZH9) was grown in the field. Strong stems were cut off at the position 50 cm away from the growing end and re-planted either in the field or in pots as stocks. Seeds of mungbean (Vigna radiata (L.) Wilczek) cv. Xiaohuijiao (XHJ) were germinated in moistened soil. Young seedlings with the hypocotyls and radicles 5-7 cm in length were harvested for making scions.

For inserting the mungbean seedling into the stem of sweet potato, we pricked a hole on the stem at the position 2-3 cm ascending the soil surface with a tapering bamboo stick. A mungbean seedling, after its radicle cut off, was inserted into the hole (Fig. 1 a, b, c). Then, the whole seedling was covered with moistened soil and shadowed immediately. The soil and shadowing were removed 3 and 7 days later, respectively.

1.2 Selection of genetic variations

Seeds of the mungbean scions were produced by selfing and harvested. They were then sowed normally in the field. We harvested the selfed seeds of the first generation (G1) and sowed them again. The same work (insemination and producing seeds) was repeated for several generations. Plants appeared with changed trait (s) were marked through generations. Seeds from the plants with changed trait(s) were harvested and sowed separately.

1.3 Analysis

Chromosomes of XHJ and the variations were analyzed according to the method of Chen[15]. Total DNA of sweet potato and mungbean was prepared with the methods described by Murray and Thompson. Southern and RAPD analyses were based on the routine methods

Fig. 1. Photographs showing the non-specific grafting between mungbean and sweet potato (a-e), G0-G2 seeds of the mungbean scion (f-h) and other non-specific grafting combinations (i-j).
    A mungbean seedling 5 cm in length (a) was used for scion after its radicles cut off (b). The scion was inserted into the stem of a sweet potato (c) and overshadowed for one week (d). G0 seeds (f, right) were harvested from the mungbean scion successfully survived on a sweet potato (e). Note that the Go seeds were slightly irregular in shape and deeper in color as compared with the normal seeds of XHJ (f, left). Normal-looking seeds (g, left) and irregular seeds (g, right) were harvested from one of the G1 plants. When the irregular seeds were sowed, "unstable seeds" (h, right) and normal-looking seeds (h, left) were produced on isolated G2 plants.
Similar non-specific grafting between mungbean and pumpkin (i) and wheat and sweet potato (j) was successful as well.

2 Results

Grafting between mungbean and sweet potato is nonspecific. A few skills are required for improving the rate of scion survival. By covering the scion with moistened soil and shadowing, 80% scions survived and showed remarkable growth on the stock. However, the scion seedling tends to generate autogenous roots from the lower hypocotyls during later growth. We cut off these roots momentarily when they were initially generated. Although the growth of the scions was largely impeded when the autogenous roots were removed completely, we usually succeeded in maintaining the survival of about 20 percent grafted scions without autogenous roots until their flowering and seeding.

With the aim of quality improvement for mungbean, we started non-specific grafting using different stocks since 1979. In the combination between mungbean and sweet potato, a large variety of inheritable variations were emerged. Fig. 2 shows the seed morphology of part of the variations. These variations were emerged from different batches of grafting done in 1978 and 1987 respectively (Table 1). All of the variations have inherited steadily for at least 20 generations. Besides the seed color and shape, life duration and the morphology of plant in some of the variations also changed distinctly (Fig. 2 and Table 1). However, these new traits did not emerge directly in the first generation. They appeared in the second to seventh generations from the "unstable seeds" (to be described below).

For showing intuitively how these varieties emerge, we repeated the process of grafting and partial selection of the variations recently with the results recorded by photographs. From a mungbean scion successfully survived on a sweet potato (Fig. 1 e), we harvested 60 seeds of mungbean (Fig. If). These seeds, comparing with the natural seeds of the scion plant, appeared no distinct changes except for they were deeper in color and relatively anomalous in shape. These minor changes in seed morphology may reflect the stressed growing state of the scion (to be discussed below). We sowed all 60 seeds in the fields. Seven of them germinated. The poor rate of germination might be caused by an occasional weather exacerbation. We numbered the plants by No. 1-7. There was no distinct change in plant morphology in this generation (G1). We harvested totally 1,116 seeds in 94 pods from the 7 plants. Among the G1 seeds, however, minor change in seed morphology was recognized in 1 pod from No. 5 (totally 17 pods harvested) and 1 pod from No. 7 (totally 18 pods harvested) plants. There were 15 seeds in each of these two pods. These seeds appeared in irregular shapes and deeper color (Fig. 1 g). We sowed the 15 irregular seeds and a few normal-looking seeds (30 seeds) harvested from No. 7 plant in the fields. Thirteen plants were generated from the irregular seeds (two seeds did not germinate). All these plants appeared normal in morphology but eight of them produced seeds with brown color and in irregular shapes (Fig. 1 h). Similar changes did not appear in the plants germinated from the normal-looking seeds.

Fig. 2. Varieties of mungbean obtained from the non-specific grafting between mungbean and sweet potato in our earlier experiments. The varieties are distinct in seed (a, XHJ; b, SL1; c, SL2; d, SL3; e, SL4; f, SL5; g, SL6 and h, SLX) and plant (i) morphologies. Note the difference between the rambling SL6 (i, left) and the erect XHJ (i, right).

 

Table 1 Partial characters of mungbean varieties obtained after grafting

Variety

Seed coat

Propensity
in growth

Thousand
grain weight (g)

First appeared
in generation

Grafted
year

XHJ

Green (dim)

Erect

40

Original scion

SL1

Yellow green

Semi-rambling

70

G3

1979

SL2

Green

Erect

70

G2

1987

SL3

Black

Semi-rambling

70

G5(G3)*

1987

SL4

Black (dim)

Semi-rambling

80

G7**

1987

SL5

Yellow brown

Erect

80

G4

1987

SL6

Green

Rambling

70

G4

1979

SLX

Brown

Erect

-

-

1987

*Black seeds with quite smaller size appeared in G3. The variety was derived after 2 generations.
** The variety was derived from SL3 after 2 more generations.

The appearance of irregular seeds with brown color was the same with the result obtained earlier. However, the seeds appeared in the first generation (G1) in 1988. Only these seeds were sowed in our earlier experiments.

A small part of the plants germinated from these seeds produced normal-looking seeds and most of them produced the same seeds. The varieties shown in Fig. 2 were emerged from the followed generations when the irregular seeds were continuously sowed. Since plants germinated from the irregular seeds with brown color produce either irregular seeds or normal-looking seeds and the varieties, we term this special kind of seeds as "unstable seeds". As an experimental control, we sowed XHJ continuously for at least 30 generations. Irregular seeds or other variations shown above were never found in the population.

For learning the possible mechanism of graft-induced variation, we first analyzed the metaphase chromosomes of the scion (XHJ) and the varieties (SL1-SL6 and SLX) obtained in our earlier experiment. The chromosome numbers of both the scion and all the varieties were 2n=22 and there was no karyotypic difference between the scion and the varieties. This result suggested that the variations might be due to the changes of gene at subchromosome level.

Cytoplasmic DNA of the scion (XHJ), a variety (SL6) and the stock (ZH9) was probed with mitochondrial and plastid specific DNA fragments. The results were shown in Fig. 3. The signals specific to ZH 9 did not appear in SL6 and there was no restriction fragment length polymorphism (RFLP) between XHJ and SL6. This result suggests that neither cytoplasmic DNA transformation between the scion and the stock nor evident recombination of cytoplasmic DNA occurs in accompany with the appearance of the variety.

Fig. 3. Southern analysis of plastid (a) and mitochondrial (b) DNA in ZH9 (S), XHJ (J) and SL6 (6). Total DNA digested with BamH I , EcoR I , Hind III and Xba I was probed with a 19.0 kb rice plastid DNA fragment, B1[18] and a 2.0 kb rice mitochondrial DNA fragment containing the full encoding region of cox I gene[19]
Fig.4. Examples of RAPD analysis of ZH9 (S), XHJ (J) and SL6 (6) DNA. Primers amplified exactly the same bond pattern in XHJ and SL6 (a, primer G13), one excess band in XH.J (b, primer C18), two excess bands in SL6 (c, primer Y18) and excess bands in SL6 with corresponding bonds in ZH9 (d, primer H8; e, primer C8) were shown, respectively. Arrowheads indicate the excess bands.

The nuclear DNA of XHJ, SL6 and ZH9 was analyzed by RAPD examination. We used 205 random primers (OPERON primers A-J and some of Ys) in our experiment. Among these primers, 5% (10 primers gave amplified signal from neither XHJ nor SL6, 37% 76 primers) gave signals without difference between XHJ and SL6, and up to 58% (119 primers) gave repeatable odd bonds between XHJ and SL6. Figure 4 shows a few examples of our result. Among the 119 primers that gave difference between XHJ and SL6, thirteen primers amplified excess bonds in SL6 and the corresponding bonds in ZH9 (see Fig. 4 for examples). We recovered DNA from these bonds for the probes in Southern analysis. The result, however, showed that none of them was homologous with those of ZH9 (data not shown).

3 Discussion

As described above, we succeeded in the inter-family grafting between mungbean and sweet potato with careful maintenance. When the seeds harvested from the scion were continuously sowed, we obtained several genetic varieties with improved quality and higher yield from the progenies. Besides the combination between mungbean and sweet potato, we also succeeded in those between mungbean and pumpkin, and wheat and sweet potato with the same method (see Fig. 11 and J for the settled scions). Genetic varieties with distinctly improved traits were obtained as well from these later combinations (data not shown).

However, the molecular mechanism for the emergence of the genetic varieties is unknown. In the present study, we analysed the alternations of cytoplasmic and nuclear DNA upon the mungbean variety. The results showed no RFLP in the cytoplasmic DNA but rich RAPD dimorphism in the nuclear DNA between a variety (SL6) and the original scion (XHJ). Traditional studies have presumed strongly that grafting-induced genetic variation may be caused by direct gene transmission from stock to scion[4, 10]. However, in our examinations, though a few PCR fragments amplified from the variety (SL6) corresponded to those amplified from the stock (sweet potato Fig. 4), there was no hybridization between the fragments when examined with Southern analyses. This result reduces the possibility of the transgenic mechanism at least in nonspecific graft system. Furthermore, although large molecules with the size up to mRNA are found to be moveable from stock to scion[20-22], there is so far no cellular or molecular evidence revealing the possibility or exact mechanism of long distance movement of DNA fragments in the graft system.

Recently, it has been indicated that environmental stress, such as tissue culture conditions activate plant transposons[23, 24]. It has been also suggested that transposition in response to environmental stress is a common phenomenon by which plants acquire genetic diversity[25, 26]. In our system of non-specific grafting, the mungbean scion is exposed to nutrient and hormone stresses throughout the growth. A mature plant of the scion species (XHJ) normally reaches 40 cm in height and produces 540 seeds on average. However, it stops growing at 20 cm in height and fruits less than 80 seeds when grafted. This large inhibition of growth and fruitage may reflect the great stress that the scion receives during non-specific grafting. If large amounts of transposition occurred during grafting, genetic variation and RAPD dimorphism as described in the present study may be explained since plant genomes may contain large amounts of transposons. In a bean species Vicia faba, for example, Ty1-copia elements may build up 1/4 of the genome.[26] Therefore, non-specific graft induced genetic variation is more likely caused by transposition rather than stock-toscion transformation. Our investigations of the transposons in the scion and the variant mungbean are in progress.

Although the exact mechanism remains unknown at the present stage, the fact that graft induces genetic variation should not be ignored in botanical research. In our experiments, genetic traits that have never been seen in the natural population of scion species emerged after nonspecific grafting (such as mungbean with black seed coat or rambling plant and some traits in a wheat variation with data not shown). Such traits can be hardly acquired from traditional cross breeding. Therefore, further studies on the application of the technique may contribute to a new way for plant breeding, while the mechanism investigation may lead to a better understanding of plant genome.

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