The AnM technique has been adopted in peanut production and proved effective in improvements of yield and disease resistance. However, the detailed mechanisms for the AnM technique have not been clarified. Therefore, in the present research, several experiments were carried out and biological fundamentals for the growth and yield improvement were examined in addition to confirmation of the agronomical advantages. Moreover, experiments were also conducted to confirm the feasibility of a modified AnM in combination with film mulching and an alternative by transplanting seedlings with extra-elongated hypocotyls, which were suggested easily practiced with mechanization.

The AnM practices included three steps. The three letters, A, n and M, showed the shapes of the section-cross of the three steps at different growth stages of the peanut crop. First, the peanut seeds were sown a little deeper than usual, about 8 cm, in the ridge to induce the extra-elongation of the hypocotyl. When the seeds were sown, the cross-section of the ridge looked like the letter A. The second, the hypocotyls elongated more than usual were exposed to light and dry air by removing the soil away around the young seedlings just after the emergence. At this time, the cross-section looked like the letter "n". The third, at the middle growth stages, soils on the both sides of ridge were earthed up to welcome the late pegs. At this time, the cross-section of the ridge looked like the letter "M".

Botanically, the elongation of peanut hypocotyl stops when the cotyledons are sent out of the soil and meet light. However, in most cases, the hypocotyl even stops elongation and leaves the cotyledon node under the soil surface when cotyledons meet light through the soil cracks. Therefore, the flowers on the early two branches from the cotyledon nodes pollinate themselves even under soil surface and produce pods earlier and the early pods compete for nutrition with the young seedlings. Therefore, the key point of "A" step was to lift up the cotyledon nodes out of the soil. At "n" step, soil around the seedling is removed and hypocotyl is exposed to light and dry air. This practice makes early pegs farther from the soil surface and thus the early formation of pods is avoided. Actually, exposing the hypocotyl is the key point of the "n" step and is also the key point of the whole AnM techniques.

In practice the modified AnM, the seeded hole on the surface of plastic films on the ridge is covered with 5-7 cm high of soil mound to induce more elongation of the hypocotyls. The soil on the film is removed after the cotyledon node is sent out of the film surface with the extra-elongated hypocotyl exposed to light and dry air. As an alternative of AnM technique more easily practiced, transplanting of seedlings was tried. Peanut seeds were first sown in seedling packs and placed in dark inside an incubator, where extra-elongation of the hypocotyls was induced. When the hypocotyls elongated to about 5 cm long, seedling packs were moved to a lighting growth chamber. The seedlings were transplanted into the field with the hypocotyls half above the soil surface.

Agronomically, all the three types of AnM practices improved plant biomass production and final shell yield with the disease resistance also increased. The yield increment was 19.2%, 16.7% and 18.9%, respectively, for the basic, modified and alternative AnM techniques. The additive and/or synergistic yield increment reached 71.2% for the modified AnM in combination with film mulching and 33.6% for AnM treatment in addition to seedling transplanting. The bio-degradable black film was better than the transparent film because the former depressed weeds and promoted soil nutrient mineralization more effectively.

Physiologically, all the three types of AnM techniques induced osmotic adjustment, which improved photosynthetic activities by maintaining a higher leaf turgor potential, especially after the hypocotyl exposing stimulation is released. The improved photosynthetic activities also reflected in less hysteresis of photosynthesis and more sensitive stomata oscillation. In the leaves of the hypocotyl-exposed peanut seedlings, stomata closed more completely when water shortage was perceived and tried to open again when leaf water was in a relative balance. The osmotic potential and leaf relative water content at zero turgor (πIP and ζIP) were lower in leaves of peanut plants with hypocotyl exposure treatment, suggesting that leaf turgor could be maintained to severer desiccation and contribute to stress resistance. Increased cell osmotic concentration might ensure the inward flow of water from apoplast to symplast and consequently the symplastic water fraction (ζsym) was larger in leaves of hypocotyl-exposed peanut plants, which might ensure, at least in part, the higher biochemical and physiological activities.

The stimulation of exposing hypocotyl to light at the "n" stage induced the production of superoxide radicals (O2・-) and the O2・- producing rate in hypocotyls and leaves of hypocotyl-exposed peanut seedlings was significantly higher than in control. The concentration of MDA in both hypocotyl and leaf was not higher, sometimes lower in hypocotyl-exposed seedlings than in control seedlings. No increase in MDA suggested that there was no real or severe oxidative damages occurred in hypocotyl exposed peanut seedlings although the superoxide radicals were really increased. This suggested that the hypocotyl exposure treatment was just a mild stimulation instead a severe stress. The SOD activity in hypocotyls and leaves of hypocotyl-exposed peanut seedlings increased in response to the stimulation of hypocotyl exposure but POD and CAT activity were not enhanced at the early period of the exposure treatment. It was suggested that exposing hypocotyl was not a stress severe enough to induce immediate activation of POD and CAT.

Soon after the hypocotyl exposure started, anthocyanin accumulation was observed visually in hypocotyls of the young seedlings. The anthocyanin accumulation is accompanied by accumulations of soluble sugars, soluble proteins as well as the production of superoxide radicals and activation of antioxidant enzymes. The microscopic observation showed that amyloplasts were fewer in the exposed hypocotyls than in the hypocotyls grown underground. Destructive consumption of carbohydrates might occur and turn into sugar or other form of energy in the exposed hypocotyls since osmotic adjustment as well as anthocyanin biosynthesis was an energy consumable process, which provided better condition for growth of shoot and root, where biomass production was improved. It is suggested that all the consequences of the xerophytophysiological responses collaborated together to make the crop healthier through their individual function in plant growth and development.

Gdi-15 (Groundnut desiccation induced) gene is a stress-responsive gene in peanut plant. It showed homology to flavonoid 3-O-glucosyltransferase which involves in the last step of anthocyanin biosynthesis. Transcript level of Gdi-15 gene in hypocotyl, where the exposing stimulation was directly imposed, was enhanced showing an up-regulation expression induced by the hypocotyl exposure, which was consistent with increased accumulation of anthocyamins and other osmotically active substances such as sugars, proline and soluble proteins. However, the transcript levels of Gdi-15 gene in leaves and root of hypocotyl-exposed peanut seedlings were not activated or showed expression of a little down-regulation.

In overall, hypocotyl exposure as a stimulation did induce the up-expression of the drought responsive gene, Gdi-15, and the consequent osmotic adjustment and anthocyanin accumulation but caused no damage to the whole plant. This is the key point of applications of xerophytophyiology and signal transduction in plant production, with false, mild or temporary drought stimulation to induce positive regulations. In conclusion, as one of practices on the theory of xerophytophysiology, the AnM techniques, especially the modified AnM as well as transplanting seedling with extra-elongated hypocotyls as the alternative of AnM practice, were effective in inducing drought responsive genes and expected positive regulations in crop production.

AnM法は、落花生栽培の改良的方法として開発された。この栽培方法には3つの段階があり、それぞれの時期に畦の横断面がA、nとMの3つの文字に似ていることからAnM法と名付けられている。まず、通常より深く種を播いて、下胚軸の余分な伸長を誘導する(A段階)。次に苗基部の土を除き通常より長く伸長した下胚軸を光と乾燥した空気に曝す(n段階)。最後に、生長の中期に畦両側の土を植物に寄せて、発生した果針が早く入土できるようにさせる(M段階)。AnM法には落花生の収量増加と耐病性増強の効果があることが確認されているが、そのメカニズムは解明されていなかった。そこで本研究では、AnM法の効果に関する生物学的な根拠を解明するための解析を行った。

1章の緒論では、研究の背景、意義と目的について述べた。

2章~8章では、AnM法、フィルムマルチと組み合わせる改良AnM法、およびAnM法の代替法とする育苗移植法の3種類の栽培法について、植物学的、農学的および植物生理学的観点から、それぞれの栽培方法の効果について詳細に解析した。

落花生が発芽して、子葉が地上に出て光をあびると下胚軸の伸長は止まるが、多くの場合、子葉は土の亀裂から入る光に受け下胚軸伸長が停止するので子葉節は土の中に残される。その結果、子葉節から生じる2つの分枝に花が咲き莢ができる。このように初期に形成された莢では成長する幼植物と養分の競合がおこるため充実した種子を形成する事は困難である。したがって、A段階で重要なのは子葉節を地上に上げることであった。n段階では、幼植物基部の土を除いて下胚軸を光と乾燥空気に曝すことにより果針を土から遠ざけ、早すぎる莢形成を妨げる。この段階で下胚軸を曝すことがn段階およびAnM法で最も重要なステップであることが明らかになった。改良AnM法では、畦上のフィルムマルチの上に種子を播いた穴に盛土して下胚軸の伸長を誘導し、子葉節が地上に出た時にマルチ上の土を除き下胚軸を曝した。またAnMの代替法として育苗移植を試した。落花生の種子を育苗パックに播き暗黒下で下胚軸の徒長を誘導し、下胚軸が十分伸びた時に育苗パックを光の下に移した後に苗を圃場に移植し、下胚軸が半分露出したままで生長させた。これらいずれの栽培方法においても物質生産と莢収量が増加した。また生分解黒ビニールは透明なビニールに比べ雑草抑制効果がよく、土壌養分の無機化が進むため増収効果がより大きかった。いずれのAnM法においても、特に胚軸露出処理を解除した後に、浸透圧調整能力が向上し、葉の膨圧を高く維持することによって光合成活性が高くなることがわかった。上昇した光合成活性は光合成滞後現象(photosynthetic hysteresis)が小さく、これは気孔の開閉反応が敏感になることで説明された。下胚軸を曝した落花生の葉では水分欠乏を感知した際に気孔がより完全に閉じ、水分が十分に与えられると速やかに気孔が開いた。下胚軸を曝した落花生の葉では、膨圧がゼロになった時点の浸透ポテンシャルとその時の相対含水率はより低く、水分欠乏に対する耐性が高いことが示唆された。細胞の浸透圧が高くなると細胞壁から原型質への水の流れを引き起こし、それに応じて生理生化学反応に利する原型質の含水率が高くなった。

9章~10章では、AnM法で外環境に曝された下胚軸において生じる酵素活性の変化、アントシアニンの蓄積およびアントシアン生合成酵素をコードする遺伝子の発現応答等について解析した。

下胚軸を曝した落下生苗の胚軸と葉のいずれにおいてもSOD活性は増加したが、MDA濃度の増加はみられなかった。したがって、下胚軸の露出により活性酸素は増加するが、これは植物にとって深刻なストレスではなく、軽い刺激となることがわかった。

また、アントシアニン合成に関与する酵素タンパク質をコードするGdi-15遺伝子の転写は胚軸露出によって促進された。胚軸露出処理後のアントシアニンは蓄積は、肉眼および定量的に確かめた。アントシアニンの蓄積とともに、糖、プロリンおよび水溶性タンパク質も蓄積した。これらの含有量の変化が、AnM法栽培における植物の健全な生長と発育に関与すると考えられた。また、細胞内の澱粉粒の蓄積が光に曝された下胚軸では少ないことから、露出処理を受けた胚軸では炭水化物が消費され、物質生産改善の一助となったことが示唆された。

以上のように、本研究では落花生栽培におけるAnM法の効果について、農学的、生理生化学的に初めて詳細に解析を行い、その有効性の要因について興味深い知見を得た。本研究で得られた成果は、今後落花生栽培方法を改良するうえで重要な情報となるため、学術上、応用上貢献することが少なくない。よって審査委員一同は本論文が博士(農学)の学位論文として価値あるものと認めた。