Mechanism Of In Vitro Morphogenesis Of Konjac (Amorphophallus Blume) And The Techniques For Its Propagation

Up to now, konjac is the only higher plant of vegetable kingdom that largely synthesizes konjac glucomannan, an importantly industrial material. Konjac is perennial herbaceous species belonging to Amorphophallus Blume (Araceae) and widely distributed throughout the mountainous areas in Southwest China with a long history of cultivation and utilization. Konjac glucomannan, a high-molecular-weight carbohydrate, is main storage substance in konjac corm. Due to its excellent physico-chemical characteristics, such as glue dissolving, gelling, thickening, film forming and mixing with other plant glues, konjac glucomannan has high values and is used widely in the food, chemistry, medicine and health protecting, and environment protection.Konjac is a one-leaf plant with a single petiole and conventionally propagated through rhizomes produced by the main corms with a low propagation coefficient. In addition, continuous asexual reproduction accumulates pathogens in corms that causes character degeneration and hence a dramatic loss in yield and degradation in quality. Due to lack of stable seed-corm propagation system, the quantity and quality of seed corm have been bottlenecks hampering development of konjac cultivation in a large scale. Since 1980s, many researchers have paid attention to development of plant tissue culture system for konjac propagation via using in vitro plants. However, in vitro plants had many disadvantages, such as low survival rate after transplantation, requiring extensive care of field management and incapability of being supplied year-around and long-distance deliver. Since 1990s, people have approached gradually the mechanisms of plant storage organ formation, and induced many asexually propagated organs in vitro, such as tuber, corm, bulblet, etc. Among them, the production system of potato seed tuber is most mature and has been industrialized, but little information is available on konjac corm production in vitro. Using konjac (A. riviveri and A. albus) as experimental materials, we carried out a set of researches on in vitro corm formation and propagation methods. The results as followings:1. Yong petiole was optimum explant for konjac tissue culture due to high frequency of desirable callus induction. MS + 1.0 mg/L NAA + 1.0 mg/L BA was optimum for callus induction and MS+0.5 mg/L NAA + 0.5 mg/L BA for callus proliferation and growth. During callus subculture, three types of calli were identified: I. watered, translucent; II. yellowish, loose in structure; III. pink or greenish, nodular or globular, compact in texture. On differentiation medium, except for type I callus, type II and III are capable of differentiating buds. Culture of typeâ…¢callus could produce complete plantlets via corm-like structure pathway. In specific culture conditions, the types of calli could change mutually. Histological observations showed that different types of calli composed of different types of cells, of which typeâ…¢callus was semi-organized tissue composed of slowly proliferated cells that contain starch and glucomannan granules. Moderate concentrations of cytokinins (BA, KT, ZT and TDZ) (1.0～2.0 mg/L) combined with low concentrations of NAA (0.1～1.0 mg/L) were optimum for typeâ…¢callus differentiation.2. In vitro morphogenesis of konjac occurred manily through organogenesis and adventitious bud and corm-like structure (CLS) were main pathways. Histological observations showed that cambium was absent in konjac petiole and that the cells outside vascular bundles of petiole firstly started dedifferentiation division to form callus. In vitro morphogenesis in A. rivieri occurred mainly through organogenesis while somatic embryogenesis took place under special culture conditions but with very low frequency. Most somatic embryos cannot convert to plants due high-frequency abnormality in these embryos. In vitro plants were obtained through adventitious bud or CLS pathways. The CLS derived plants had complete root systems without rooting culture while adventitious buds could not root during differentiation culture and grew slim. Both adventitious bud and CLS originated from subepidermal cells of typeâ…¢callus that firstly form meristematic masses during further differentiation culture. The meristematic masses could directly develop into bud primordia that developed further into adventitious buds. Meanwhile, the masses could convert to interim globoids that have apical meristem and showed indistinct leaf primordia and developed into CLSs and complete plantlets during further subcultures.3. Endogenous GA₃ content increased with bud formation whilst JA increased with corm development, indicating balance of endogenous hormones a key factor influencing organogenesis pathways. During the course of petiole parenchyma cells to form callus, the contents of endogenous hormones (IAA, GA₃, ABA and JA) changed obviously. Endogenous IAA level fell at initial culture stage and then rapidly rose 10d later and was stable after culture for 30d. Endogenous ABA content fell within first 20d and increased during further culture. Endogenous GA₃ and JA content decreased during the whole induction culture. The alternation of contents of the three endogenous hormones (GA₃, ABA and JA), either in the callus that formed CLSs or in the one that differentiated adventitious buds, showed different tendencies. The content of endogenous GA₃ hardly changed in CLS organogenesis but rapidly increased in adventitious bud organogenesis. Endogenous ABA level was almost stable during CLS organogenesis but rapidly fell in adventitious bud organogenesis pathway. Endogenous JA level rose obviously in CLS formation but showed an indistinct change in adventitious bud differentiation. Only the content of endogenous IAA decreased consistently in both the two organogenesis pathways. The balance of GA3 to ABA or JA showed different patterns in the two pathways, but the latter one showed most remarkable difference. The GA₃/JA ratio showed completely reversed tendencies in the two pathways. These results indicated that the pathways of konjac organogenesis in vitro were related to the changes of endogenous hormones and their balances.4. In vitro konjac corm formation was influenced by medium and culture condition. The combinations of BA at moderate concentrations (1.0～2.0 mg/L) and NAA at low concentration (0.1～0.5 rag/L) favoured corm formation from type III callus. Among these combinations, MS+2.0 mg/L BA+0.5 mg/L NAA was most effective for both A. rivieri and A. albus. Increase of sucrose concentrations in a certain range (2-8%) favoured A. albus corm formation; 6% (w/v) was most efficient. As for A. rivieri, lower concentration (4%) was required for corm formation. In vitro corm formation was not affected by light periods but greatly influenced by culture temperature. Cultured in 22℃, the percentage of corm formation, mean number of corms and mean corm fresh weight reached their maximum. Wounding the apical meristem of the corm could overcome apical dominance and promote cormel propagation.5. Selection and culture of konjac callus favoured lowering the genetic variation frequency of plants regenerated from the callus. Four groups of regenerated plants GO (derived from the first culture), G2 (derived from the third subculture), G4 (derived from the fifth subculture) and G7 (derived from the eighth subculture) were obtained by selection and culture of well-developed typeâ…¢callus. RAFD and ISSR analyses showed that the genetic variation frequency of G0 was the highest (RAPD: 15.6%; ISSR: 18.1%) followed by G2, G4 and G7. Among the four groups, GO had more variation loci (RAPD) than those of the other three. The variation loci detected by ISSR were more than those detected by RAFD and distributed randomly throughout the four groups. From these results, selective culture of typeâ…¢callus provided a good measure to control genetic variation of their regenerated plants. The results revealed that the genetic instability induced by konjac tissue culture mainly took place in repeat regions of konjac genome, which hardly results in morphological variation.6. In vitro corm system is a suitable way applicable to konjac propagation. In vitro corm had obvious advantages over in vitro plants in adaptability under field conditions. The growth of both in vitro corm and plantlets was compared in the field and the results revealed that although both in vitro derived plants and corms could produce corms after transplantation the survival rate of in vitro plants was much affected by temperature during transplanting. However, in vitro corm grew vigorously and had high survival rate, particularly when the large corms (over 0.5g in weight) were used and the physiological dormancy was broken. The harvested corms produced by in vitro corms were larger in size than those produced by plantlets and hence more suitable for commercial propagation of seed corms.