Inheritance Of A No Vine-type Cucurbita Moschata And Establishment Of Its High Frequency Regeneration System

There are three economically important Cucurbita species, namely, C. pepo L., C. maxima Duch. and C. moschata Duch., which have different climatic adaptations and are widely distributed in agricultural regions worldwide. C. moschata plants possess long internodes and have trailing vines that may spread up to 15 m from the crown of the plants, thereby requiring wide planting distances. Some plants of developed cultivars of C. pepo L have short internodes. No vine plants in C. moschata have some features that make them valuable, i.e. they allow higher plant densities, facilitate cultivation, and have a more concentrated maturity than vine counterparts. This makes them value in pumpkin breeding. Curcubita is affected worldwide by several viral diseases that substantially reduce yield and fruit quality. Plant biotechnology appears to be a viable option for the improvement of the Cucurbita species by means of plant tissue culture and genetic transformation. The establishment of an efficient in vitro plant regeneration system suitable for genetic transformation is the key step in this approach. On the other hand, it is generally known that from the factors which influence organogenesis, the foremost are genotype, age and origin of the initial tissues, which, in turn, are related to their endogenous hormonal status. Moreover, the importance of the exogenous application of plant growth regulators is much discussed. In this wise, the organogenic potential of explants is closely associated with their contents of natural and exogenous applied plant growth regulators.The purposes of the present work were to study the morphology and inheritance of the no vine pumpkin and establish a reproducible organogenic pathway for C. moschata. On this condition, we studied the correlation between the organogenic capacity of pumpkin cotyledon explants, as a function of their developmental stage, and endogenous hormonal contents. The main steps are as follows:(1) The no vine plants differ greatly in morphology from vine plants. No vine plants could readily be identified by its vine characteristics when three true leaves were present. No vine plants had short internodes, short vine and small number of internode compared with vine plants. Vine plants produced more male flowers (21.1±4.3) than no vine plants (13.7±3.3). In contrast, no vine plants flowered eight days earlier than vine plants. The morphology of F₁ plants is also investigated. F₁ plants of crosses between no vine and vine resembled vegetatively the bush parent. Although no vine and F₁ plants had a higher rate of fruit abortion than vine plants, they had a larger total number of fruit than vine plants in a plant basis. All F₁ progeny were no vine phenotype regardless of the direction of crosses. The reciprocal BC₁ families were segregated approximately one no vine: one vine plant. In F₂ the distribution was 156 no vine to 49 vine-type, which fitted a 3:1 ratio.(2) To induce multiple shoots from pumpkin, cotyledon explants excised from various ages of seedlings after germination in vitro were cultured on MS medium augmented with different concentrations of BA. Highest frequency shoot regeneration (63.7%) was induced from 7-day-old cotyledon cultured on MS medium containing 0.5 mg·L^-1 BA. The frequency and time of shoot induction had closed correlation with donor seedling age. Multiple shoots elongated on MS medium with 0.1 mg·L^-1 BA and 5～7 shoots per explant were recovered. The number of shoot per explant had no relationship with donor seedling age or BA concentration of induction medium. Elongated shoots were rooted on MS medium easier than on 2/3MS, 1/2MS or MS supplemented with 0.1 mg·L^-1 NAA. The rooted shoots were then transferred to greenhouse where they grew and flowered normally.(3) Quantitative analysis of endogenous cytokinins (ZR and iPA) and auxin (IAA) in initial cotyledon explants of different ages showed that cotyledon explants with the highest caulogenic potential in each BAP concentration had the highest contents of iPA and Cks/IAA ratio, and that with the lowest caulogenic potential in each BA concentration had the lowest contents of iPA and Cks/IAA ratio. These results also suggested that the direct organogenesis of pumpkin was characterized by changes in hormone levels. A general principle that a high concentration of cytokinins and a low concentration of auxin promote the induction of shoot morphogenesis in vitro was confirmed in our studies.In conclusion, the morphology of vine and bush plants in C. moschata is compared in this report. Genetic analysis of plant habit shows that a monogenic inheritance is expressed whereas no vine is dominant to vine type. On the other hand, an effective regeneration protocol was constructed for C. moschata, though only one cultivar was tested. On the basis of endogenous analysis, we attributed a major role to iPA and the ration of IAA/Cks (ZR and iPA) in caulogenic response of pumpkin cotyledon explants.