"In Vitro Introduction, Proliferation and Medium-Term Establishment of Various Yams’ Species (Dioscorea Spp.)"

Series of experiments were conducted in this study. The experiments begin with the use of yams’ aerial tuber as an explant for in-vitro introduction. Small aerial tubers of two yam species, obtained from 9 months old plants grown in a greenhouse were sterilized and cultivated in three different media. D. polystachya Turcz. gave 68% regeneration of shoots and roots which was significantly higher than that of D. bulbifera L. (16%) 4 weeks after culture. In the second experiment, studies went on an already existing in vitro collection to determine the multiplication rate during storage. Thirty-five yam accessions, established in two different temperature regimes, were used to determine the multiplication rate by sub-culturing sin-gle nodes with one leaf every two months. Sub-tropical/temperate yams theoretically can multiply almost as good as tropical yams. However, not all explants can further multiply. In the third experiment, determination of TMR was continued on five selected genotypes representing four different species up to 10 cycles of sub-culture. D. bilbufera, D. polystachya and D. cayanensis Lam., exhibited consistent theoretical multiplication rate whilst the multi-plication rate for D. alata L. decreased at the initial study but started to recover after 4 cycles of sub-culture. Received: July 14, 2021


Introduction
There exist about 600 species of yams (Dioscorea spp.) in the world belonging to 23 sections [1]. Some of the species have been cultivated as food crops for centuries because of the high carbohydrate content in their tubers and some others have been used as medicinal plants due to the presence of chemical compounds (mainly diosgenin) which have been used to produce steroid drugs.
The main food crop species are vegetatively propagated; therefore, preservation of yams can be accomplished through in vitro short and medium-term conservation and cryopreservation.
Successful introduction of yams into in vitro culture followed by their multiplication would provide sufficient explants for various needs including experiments on germplasm preservation.
Introducing yams into in vitro culture has been done using various explants such as shoot and node culture, seed culture (in case of wild species) as well as tuber culture using various culture media [2][3][4][5][6][7]. Information on multiplication rates of various accessions is important for their maintenance and other practical uses. It is also desirable that all genetic potential of the preserved germplasm, including multiplication rate, be maintained or stable in the long run. However, reports have indicated the occurrences of genetic changes resulting in soma-clonal variation in the in vitro culture (see Scowcroft, 1984) [8]. Evaluation of long-term multiplication rates is, therefore, important to ensure that the in vitro cultural

In vitro Introduction
Experiment on the in vitro introduction was conducted during Tubers were washed with water and then with 70% alcohol before being sterilized using 20% sodium hypochlorite plus 2 drops of Tween 20 on a shaker for 15 minutes. The tubers were than washed four times with sterile water before the introduction into culture.
Samples of Yam 21 were mostly cultured as an entire tuber (cut into two only when necessary) while many of the tubers of Yam 16 were cut longitudinally into two (head is divided into two parts) before being cultivated in 15 ml 'introduction' medium in SIGMA glass culture tubes (length 15 cm, diameter 2.5 cm). Three different media were tested, namely I1 [9] salts + 0.1 mg/l thiamine + 2 mg/l indole acetic acid [IAA] + 5 mg/l kinetin), I2 (MS salts + 0.1 mg/l thiamine + 2 mg/l IAA + 10 mg/l kinetin), and I3 (MS salts + MS vitamins + 0.2 mg/l α-naphthylacetic acid [NAA] + 0.5 mg/l 6-benzylaminopurine [BAP]). All media used 3% sucrose, 1% agar and 0.2 % activated charcoal (AC). Each treatment combination was replicated two times by using two culture racks each consisting of 18 culture tubes. The two racks of each treatment combination were placed on different shelves in the growth chamber with 16h/8h light/dark period using fluorescent lamps, 25°C and light intensity of 60-80 µmol.cm-2 . s -1 . Observation was conducted during four weeks on the percentage of shoot and roots production, callus formation and the explants which showed no reaction as well as infected explants (infected explants were directly discarded from the growth chamber and were not used in calculating the percentage of each parameter). Percentages of the explants produced only roots as well as roots and callus were also noted, however, were not presented in the results due to their very small number.

Multiplication Rate
Observation on multiplication rates involved 46 clones comprising 39 accessions and 18 different species of a research collection maintained in IPK. The materials were obtained from various sources such as botanic gardens or brought by students from other countries either as in vitro plantlets, tubers or seeds. Most of the genotypes were introduced into culture via in vitro sowing as well as using nodal explants of plants grown in the greenhouse.
Most of the collections have been maintained since December 1999 by means of sub-culturing single nodal explants (with one small leaf) every two months in 15 ml of MS medium + 0.1 mg/l NAA + 2 mg/l BAP with 3% sucrose, 1% agar and 0.2% AC in SIGMA glass culture tubes (length 15 cm, diameter 2.5 cm). Previously, culture environment used was a 25 °C growth room with 16 h photoperiod

Statistical Analysis
Percentage of shoots and roots production of the experiment on in vitro introduction was subjected to ANOVA to determine the main effect and the interaction between medium and genotype. The t-test was used to assess the differences on TMR and PMR mean values among all genotypes as well as between TMR and PMR at each genotype. For study on the medium-term TMR, Pearson correlation coefficient was used to assess the relationship be-tween variables (TMR and number of sub-culture) on each genotype. Furthermore, polynomial regression analysis was implemented to estimate the curve, which most fit to the trend of the data. All the data were tested for normality distribution and equal variance before analysis. The analyses were conducted with the help of SigmaStat software, Version 2.0, SPSS Inc., Chicago.

In Vitro Introduction
Germinated explants were on various stages of development after four weeks of cultivation. The latest stage of development for the two species are given in Figure 1 (taken 5 weeks after culture).
ANOVA was conducted only for the formation of shoots and roots (germinated explants) since they were considered to be the most important developmental character for the in vitro introduction.
The statistical analysis indicated that there was no interaction between genotype and 'introduction medium' used. The main effects of each treatment, therefore, discussed. Genotype effect: The fact that Yam 16 was able to produce shoots and roots in this experi-ment (Table 1)  In the present experiment, Yam 21 germinated faster and produced significantly higher shoots and roots compared to Yam 16 ( Table   1). Special morphological characteristics of tuber seemed to be one of the factors re-sponsible for these differences. D. bulbifera has a trait of a smooth tuber surface while the tuber of D. polystachya has many papillae on its surface from which germination can take place, although, in most cases, germination occurs on the head of the tuber. In the previous in vitro introduction using Yam 25 (D. sansibarensis Pax) which also possesses many papillas on the tuber surface, we were able to obtain 28% germination (shoots and   Note: Contaminated explants were not included in calculating percentages of different developmental characters. §) Parameter analysed statistically. *) Numbers within the column below genotype followed by similar letter are not significantly different at the 5% level based on F-Test. **) Numbers within the column below medium followed by similar letter are not significantly different at the 5% level based on the F-Test.

Medium Effect
Although medium I1 showed slightly better effect (50.8%) on the production of shoots and roots compared to that of I 2 (42.2%) and I3 (35.0%), this effect was statistically not significant ( Table   1) The percentage of non-responsive explants was slightly higher in medium I3 than in the other two media (Table 1).

Short-Term Multiplication Rates
It has been indicated that grouping the accessions in two culture with more than one clone, and four of these clones were lost in course of the maintenance. For some accessions, two clones were used in calculating multiplication rates. The second intro-duction of these accessions was considered as replication ( Table 2). Note: Numbers (values±standard errors) within the column (between accessions) followed by the same letter (s) are not significantly different at α=0.05 based on t-test. **) Numbers (values±standard errors) within the row (within accession) followed by the same letter (s) are not significantly different at α =0.05 based on t-tes, BG = Botanical garden, C= contaminated explants, NS = Not survived explants, TMR = theoretical multiplication rate, PMR = practical multiplication rate, R = replication, T = temperature.

Medium-Term Multiplication Rate
Generally, correlation coefficient (r) indicated a decrease

Discussion
Traditionally, genetic conservation of vegetatively propagated crops including Dioscorea spp. has been done through field cultivation. Field maintenance, however, cannot guarantee the security of collection, since the loss can occur through pests and diseases, virus, poor sprouting, unfavorable storage conditions, drought and poor handling, which has been reported for yams to achieve 10% annually [11]. Beside this, it is also time and space consuming and laborious. In vitro gene bank, therefore, offers a better way of conserving genetic diversity of such crops. To be able to conserve yams germplasm through in vitro culture using slow growth conditions and cryopreservation, as well as for other purposes such as rapid multiplication, producing virus free plantlets through meristem culture, and germplasm exchange, in vitro introduction would be the first step. This has to be followed by the establishment of the cultures for which information on the multiplication rates of the introduced clones is important.
Previous research has shown that in vitro sowing gave the highest rate of success followed by introduction of nodal cuttings [5]. But yam (Dioscorea spp.) does not always produce seeds in the field [12]. Meristem tips have also been used with high success as primary explants. It takes, however, slightly longer periods (28 weeks) to produce plantlets [3]. Attempts to introduce yams using underground tubers have not been successfully done, since all the cultures became necrotic within eight weeks of culture [3].
Our experiment indicated that yam can be introduced into in vitro culture by means of aerial tuber in a quite short time and that cultivating whole tuber of small size would be more suitable than sets of cut tuber, which may result in high contamination and callus production.
The experiment also evaluated three different culture media which have been used for introduction and establishment (with slight modification). In I1 and I2, different concentrations of kinetin combined with IAA were used to supplement MS basal medium [6], while in I3, BAP and NAA were used to supplement MS medium [7]. The results indicated that there was no statistical difference between these three media, although I1 medium gave slightly greater effect, followed by I2 and I3, respectively. This finding was in contrast with that of Mitchell et al. (1995a) [3], who reported that D. cayenensis, D. trifida and D. rotundata were more responsive to BAP than to kinetin for shoot production. This may, however, be explained by lower concentrations of BAP and NAA used in our case. Furthermore, the species and materials used in these studies were different.
Information on multiplication rates is not only important for commercial purposes of rapid multiplication. It is also useful to support decisions of whether certain measurements should be taken in case of a given accession for its safe germplasm preservation.
Theoretical multiplication rate represents genetic potential of the accession to reproduce in vitro in the next subculture. Practical multiplication rate, on the other hand, shows environmental influences on the genetic potential which in this case including selection of the explants, culture media used, sterility of culture environment and the technical work of sub-culturing.
Several studies have been conducted on multiplication rates of yams [3,4,6]. Those studies, however, were focused only on some edible, tropical yams and on the theoretical multiplication rate.
TMR shown by D. cayenensis (Yam 45) in our experiment was higher  [4]. For D. trifida 'Short Neck Yampie', however, they were able to obtain a multiplication rate of 5.0 after 4 weeks of subculture on basal medium supplemented with 0.5 mg/l BAP using nodal explants initiated from small tubers which was higher than our finding on D. trifida 'Yampie' of 3.68±1.91 (Table   2). These differences may be attributable to different media used and different methods of determining the MR (TMR). Mitchell et al. cayenensis and D. trifida. They found that older nodes from plantlets grown in vitro, regardless of their origin, produced more shoots.
Younger nodes grew weaker, but there was more swelling and bud development on nodal regions which, in the next subculture, resulted in higher growth. For the sub-tropical/temperate yams possessing more woody stem, however, not all explants can be propagated upon culture.
Monitoring genetic stability during in vitro germplasm maintenance is considered important due to the possible genetic abnormalities affecting the trueness-to type of the preserved materials. This has been implemented in various in vitro gene banks in the world by evaluating morphological, cytologycal, biochemical and DNA (RNA) characteristics [13][14][15]. In the context of further studies in yams germplasm conservation including cryopreservation, in which a great number of explants are needed, consistent production of number of shoots, nodes and branches (proliferation) enable a determination of an efficient number of plantlets to be maintained and/or multiplied for a continuously running experiment.

Conclusion
This study demonstrated that aerial tubers of yams (Dioscorea spp) can be used as source materials to be introduced into in vitro culture. Yams originated and distributed in sub-tropical and temperate areas are able to produce almost as many explants as tropical yams. However, not all of these explants can reproduce in the next subculture on the culture medium used for this study. For these accessions, therefore, care should be taken on the selection of explants, and further research should be done aiming at improving medium and other conditions for their multiplication. For tropical yams, on the other hand, every node explants regardless of their position is able to reproduce upon subculture. TMR was consistent after 10 cycles of sub-culture on four of five genotypes studied; one genotype showed reduction of TMR at initial cycles of sub-culture but recovered after 4 cycles.