2. Conservation of Mulberry Genetic Resources in Japan

2.1 Present status of germplasm maintenance

2.1.1 Mulberry Species maintained at NIAS

We have adopted the classification system of the mulberry species which was established by Koizumi (1917), though many scholars classified mulberry species using different methods.

According to Koizumi’s system (classified the genus Morus into 24 species and one subspecies), we classified the mulberry accessions preserved at NIAS into 22 species, which includes M. boninensis K., a very rare species indigenous to Japan and has been registered in the red data book as a critically endangered mulberry species.

The extinction of M. boninensis is due to the cross contamination with M. acidosa Griff. which has been introduced artificially from Okinawa islands (Koyama et al., 1998).

2.1.2 Origin of Mulberry species

Genetic resources of mulberry are classified into five groups based on their origin: wild type, domestic type, bred type, others and unknown. The number of genotypes belonging to each group is as follows:

2.1.3 The place of origin

The number of genotypes classified in accordance with the places of origin is as follows:

2.1.4 Ploidy level in mulberry

A lot of domestic, natural triploid varieties, such as “Ichibei”, “Fukushima ooha”, “Akagi” and “Tagowase”, and five artificial triploid varieties, “Shinkenmochi”, “Aobanezumi”, “Mitsushigeri”, “Yukimasari” and “Yukiasahi” have been preserved in NIAS. Both M. serrata Roxb., indigenous to India, and M. tiliaefolia Makino, originally from Japan and Korea, which are known to be hexaploid, are kept as accessions.

We have preserved eight genotypes of M. boninensis Koidz., which are natural tetraploid. M. nigra L. which is dexoploid (2n=308), has been maintained as one of the most valuable accession among mulberry genetic resources. Twenty-two strains of artificial tetraploids have been developed from practical varieties, and are applied for mother plant to breed new triploid cultivars.

2.1.5 Classification of the existing germplasm

Genetic resources of mulberry are classified into three groups: working collection, base collection and active collection. The genotypes of the working collection are taken into account during in field examination and investigation. The genotypes classified into base collection are genotypes that are necessary to preserve for a long term as a result of the investigation. Genetic resources that are judged to distribute out of base collection are grouped as active collection. Almost all genotypes belonging to active collection are domestic varieties. In principle, it is difficult to distribute new developed cultivars or exotic genotypes to the foreign countries. At present, 430 genotypes belong to working collection and 945 genotypes including 780 genotypes of active collection are base collection.

2.1.6 Encountering challenges and their solutions

There are several challenges encountered at present to maintain and conserve the mulberry genetic resources. The number of researchers being involved in mulberry management has dramatically been reduced to only one after the reorganization of NISES in April, 2000. The lack of working force will eventually hamper the management and renewal of mulberry data.

The consequences due to the shortage of helping hands have also escalated the disappearance of genetic resources due to the damage by the disease and pest which are difficult to control. Urgent problems are dwarf disease and long-horned beetle.

Double preservation of mulberry germplasm is not been carried out at different places in Japan. At present, the germplasm is only managed at our laboratory, though the double preservation of genetic resources had been carried at the branch of our institute till 1996. We have to initiate the execution of the double preservation to save the existing germplasm which is already at the danger of dispersion, and remains a difficult challenge for us.

2.2 Management and utilization of germplasm resources

2.2.1 Methods employed in preservation

Mulberry genetic resources are preserved by following methods: field cultivation, pot cultivation and greenhouse cultivation. In case of field cultivation, five plants of each genotype are planted, and pruned at bottom part once a year in March (spring pruning) or in June (summer pruning). The stump is maintained by low-cut training method, and spacing is 2.5m x 0.6m. Control of pest and disease are being carried out routinely. New saplings are planted to compensate for the stumps which are withered to death because of unknown reasons. In Japan, propagation of mulberry is generally carried out by grafting methods, because most of the domestic genotypes have poor rooting ability. In the case of exotic accessions, however, a lot of genotypes show good rooting ability.

Genotypes which lack resistance to pest/disease or show weak growth are preserved as potted plants. Almost genotypes of non-dormancy type, such as the accessions introduced from Southeast Asia, are managed in the greenhouse, because they can not survive at field under winter condition in Japan.

2.2.2 Utilization of germplasm resources

The accession which is classified as working collection can be distributed as an experimental material. The price is 5,700 yen with 1 unit (3 nursery trees or 3 branches for grafting). Total 90 units have been distributed all over Japan in the past five years. Distributed genetic resources were utilized effectively and showed good results.

To utilize genetic resources efficiently in mulberry breeding programs, morphological traits necessary for classification and identification, agronomical traits involved in the resistance against diseases and pests, and commercial traits related to productivity and quality have been investigated for a long time. Detailed information on the morphological and agronomical traits of about 260 genotypes has already been obtained and published (Kobayashi et al., 1994). The traits were moderately examined according to the investigation method as described previously by Machii et al. (1997).

2.3 Development of germplasm resources

Excellent results have been produced by development of germplasm resources in Japan. We will introduce some examples here.

2.3.1 Development of a new cultivar suitable for mechanical harvesting

Mulberry was originally cultivated in Japan, as well as in other countries, for sericulture. 20 mulberry cultivars for sericulture were registered and released from 1971 to 2000 by the Ministry of Agriculture, Forestry and Fisheries. All of them were developed using the method of artificial crossing. Commercial varieties and bred strains/cultivars were used as mother plants mainly in order to breed a cultivar showing high productivity and quality. However, preserved genotypes, such as “Akamerosou” and “Murasakiwase”, has been applied as beneficial breeding sources for the development of resistant cultivars against dwarf disease, die back etc.

Breeding targets of mulberry have been to increase yield, nutritional value and resistance against diseases and pests for long time. Nowadays, new targets have been added to cope with changes in the sericultural system such as to increase the numbers of silkworm reared and their adaptability to densely planted fields suitable for mechanical harvesting. From this point of view, we developed a new cultivar “Natsunobori” making use of genetic resource effectively (Fig. 1).

“Natsunobori” was selected from the F1 generation of “Ichinose” x “No. 3001”. “No. 3001” is the developed strain belonging to M. latifolia Poir, and has been preserved in germplasm for more than forty years. This combination was aimed to develop a variety with high quality, yield and adaptability for mechanical harvesting, considering the fact that “Ichinose” shows good quality and high yield as compared to that of “No. 3001” which has thin uniformly branches and an erect tree type structure. The characters of “Natsunobori” were highly evaluated from all the concerned researchers when compared with control variety “ Shin-ichinose “.

“Natsunobori” has the following characteristics:

1. It is a diploid cultivar belonging to Morus alba L.

2. Its form is characterized by erect type resulting in tolerance to lodging.

3. Its shoots are thin, long and more in number than those of “Shin-ichinose”. The internodal distance is somewhat short.

4. “Natsunobori” has lobed, somewhat large, smooth, glossy leaves in spring to late autumn. The leaves are green and hardening is in late autumn and is nearly the same as that of “Shin-ichinose”.

5. The young shoots grow vigorously and uniformly after sprouting. Re-sprouting after intermediate pruning in summer and autumn is vigorous.

6. “Natsunobori” has the same leaf yield and quality as “Shin-ichinose” which has an established reputation of the high productivity and the quality.

7. “Natsunobori” shows the same level of resistant against dwarf disease and bacterial blight as “Shin-ichinose”. Shoot tip cold injury is less pronounced than in “Shin-ichinose”.

Overall, “Natsunobori” can be easily cultivated in warm, open areas of Japan and is adaptable to densely planted cultivation. Furthermore, its morphological character is the most suitable for mechanical harvesting in summer and spring pruning. “Natsunobori” is thus suitable for multiple silkworm rearing.

2.3.2 Development of a new cultivar suitable for fruit production

In recent years, mulberry has being re-evaluated due to its functional characteristics and is being utilized for various purposes, such as fruit, medical use, animal feed and so on. In this paper, we will introduce new mulberry cultivars for fruit production.

The mulberry fruit has recently been evaluated as one of the products that could stimulate the activity of upland farming in Japan. Products processed from mulberry fruits in food industries, such as jam and wine, are being sold in a small scale all over the country. In order to meet the demand of mulberry varieties with high fruit productivity, “Kataneo” and “Okaraguwa” were selected as genotypes suitable for fruit production from a large number of genetic mulberry resources (Machii et al., 1999). Therefore, we chose the method of polyploidy breeding to develop strains which bear larger fruits from the two selected genotypes to achieve high productivity (Koyama et al., 2001). These two strains were designated as “Lalaberry” and “Popberry” (Fig. 2, 3), and officially registered as mulberry cultivars suitable for fruit production for the first time in Japan.

“Lalaberry” and “Popberry” were bred from “Kataneo” and “Okaraguwa”, respectively, both of which were developed from a colchicine treatment. It was found that these two cultivars were mixoploids comprised of diploid and tetraploid as confirmed by chromosome analysis. Moreover, it was found that these cultivars were peripheral cytochimeras, showing orderly structure of diploid-tetraploid-tetraploid-tetraploid from the outermost cell layer to the innermost. This structure is found to be consistently stable.

Generally, it is shown that autotetraploids, such as the strains developed by colchicine treatment, make various cells and tissues gigantic. “Lalaberry” and “Popberry” possess diploid cells at the surface, but the other tissues consist of tetraploid cells because of the chimera structure, which may explain why the fruits became so large.

“Lalaberry” and “Popberry” bore many fruits in the second year after field plantation. The number of fruits was about 30% less than that of the original genotypes, however, the average weight of fruits was almost two times. The total yield of the fruits for the period of three years (three to five years after plantation), increased by about 20% in Lalaberry and about 35% in Popberry, respectively, as compared to “Kataneo” and “Okaraguwa”.

Comparing the new two cultivars, “Popberry” exceeds “Lalaberry” in size of fruits, and maximum weight exceeds over 15g per fruit. “Lalaberry” shows high production ability thereby yielding fruits about 5 kg per plant every year, which is more than that of “Popberry” (Table 1). The sugar content of fruits was about 9 Brix%, and the fruit was very juicy. Therefore, the fruit from these cultivars are considered not only suitable for processing, but can also be substitute as an edible fresh fruit.

To spend a healthy life, anthocyanins have recently been assigned to function as an anti-oxidative. It is well known that mulberry fruits contain a high concentration of anthocyanins. Moreover, it was found that 1-deoxynojirimycin, which is said to have a profound effect in lowering the blood-sugar level in diabetic patients, is amply present in mulberry fruits. Elucidation of new properties of mulberry fruits should promote their use as a healthy food. We hope that these cultivars can provide ample supply of mulberry fruits to pharmaceutical and food industries and will further promote the activity of upland farming in Japan.

2.3.3 Genetic analysis of the peculiar characters

There are several genotypes possessing peculiar characters among the germplasm in Japan (Fig. 4). They may be of vital use to biological studies. Many of them are derived from bud mutation, and shows peripheral chimera. We have demonstrated that genetically homogeneous plants can be regenerated by in vitro culture via adventitious buds induced on leaves of chimera plants. The method and results of in vitro culture are shown in Figure 5. Wild type plants were regenerated from “Kibajumonji” and “Chijimiguwa”, whereas plants with more severe characters than original one were regenerated from “Ryoumenguwa”.

We began genetic analysis of the peculiar characters associated with “Chijimiguwa” as shown in Table 2. About half of the offsprings of “Chijimiguwa” crossed with wild type were chijimiguwa-type and the rest showed wild type characters. It infers that “Chijimiguwa” has its mutation in the cells at the internal tunica layer of the meristem. These results indicated that “Chijimiguwa” is a peripheral chimera with wild-type cells (outside) and the chijimiguwa-type cells (inside). The results from the genetic analysis and in vitro culture show that intermediate type plants occurring from the offsprings were chimeric with chijimiguwa-type cells (outside) and the wild-type cells (inside). Eventually, all results of crossing and in vitro culture indicated that the peculiar characters of “Chijimiguwa” were directed by a single mutable gene, which we named as Chijimi1-1.