Induction And Differentiation Of Calluses In Anther Culture In Bitter Gourd (Momordica Charantia L.)

Bitter gourd (Momordica charantia L.), a herbaceous annual or perennial climber of the family cucurbitaceae, has been widely grown all over the world for its medical and nutritional value. As an effective assistant method for plant breeding, anther culture generates haploid which makes plant breeding more efficient and less time consuming. However, reports of anther culture in cucurbitaceous vegetables are limited and bitter gourd is now without success because of the reluctance in differentiation. In this study, four cultivars (F1) of bitter gourd (Momordica charantia L.) were used for anther culture. The main results were as follows.(1) The relationship between length of flower buds, color of anthers and developmental stage of microspores was established. 4-5mm flower buds and green-white or light green anthers were indicative of the microspores at uninucleate stage.(2) Callus induction rate of some genotypes increased after cold pretreatment to anthers (in darkness, at 4℃), while some decreased. Cold pretreatment to anthers for 2-3 days resulted in the best induction rate. Callus could hardly be induced if cold pretreatment exceeded 4 days.(3) On MS medium with 30g sucrose, different combinations of PGRs gave rise to induction rates ranging from 0 to 84.38% among four genotypes. 2, 4-D (0.1 mg·L^-1) only, low concentration of 2, 4-D (0.1 mg·L^-1) in combination with each level of 6-BA and low concentration of 6-BA (0.2 mg·L^-1) in combination with each level of 2, 4-D were unable to induce calluses. 2, 4-D was more effective than NAA in callus induction. The optimal PGR combination for callus induction was 2,4-D 0.5 mg·L^-1 + 6-BA 2.0 mg·L^-1.(4) The induction rate decreased when the concentration of sucrose ascended from 3% to 9%, while emergence of callus was delayed. Anther wall derived calluses could be inhibited when higher level of sucrose was added to the medium. 7% sucrose in medium caused a relatively higher induction rate and made anther wall derived calluses completely inhibited.(5) Anthers cultured under light resulted in lower induction rate than those cultured in darkness did but the texture of calluses were denser and more vigorous. The higher culture temperature we used, the higher induction rate we got and the looser callus texture took on. A little part of calluses from 'Pangniu', subcultured on MS supplemented with TDZ 0.5mg·L^-1,2, 4-D 0.5mg·L^-1 and 6% sucrose, gradually turned smooth and delicate. These transformed calluses were histologically proved embryogenic.(6) The callus induction rate of anthers with different flower-bud length, anther color and microspore developmental stage matched well. The induction rate of microspores at middle-uninucleate and late-uninucleate stage was about 60%, at early-binucleate and middle-binucleate stage, was about 35%.(7) Different inoculating orientation, sampling time and sampling location all had significant effects on callus induction. The optimal inoculating orientation was the way in that the anthers were planted standing-straight on medium. Anthers sampling from the main stem before June were better for culture.(8) Calluses obtained in the study could be morphologically divided into four types, typeⅠ: global, semitransparent, dense and with acicular structures on surface; typeⅡ: light yellow, semitransparent and dense; typeⅢ: white, big, tumor-like and with smooth surface; typeⅣ: fragile, more semitransparent and homogeneous (embryogenic callus).(9) Four genotypes involved in the study all failed to regenerate plantlet. 40% calluses of cv. 'Pangniu' rooted well on MS medium supplemented with 0.05 mg·L^-1 NAA and 0.1 mg·L^-1 KT. 12.5% calluses of cv. 'Jiuzacuilu' rooted on MS medium supplemented with 0.05 mg·L^-1 NAA and 0.05 mg·L^-1 ZT. Embryogenic calluses of 'Pangniu' formed smooth protuberant structures on MS medium supplemented with 0.1 mg·L^-1 NAA and 0.1 mg·L^-1 TDZ. On MS medium supplemented with 0.2 mg·L^-1 IBA and 3.0 mg·L^-1 6-BA, green protuberant structures were produced on the surface of calluses of 'Bixiu'.(10) Chromosomal examination of root tip cells revealed that, of 26 roots from 'Pangniu', 4 roots were haploids and the others were mixoploids.