Study On Tissue Culture And Tetraploid Inducement Of Balsam Pear (Momordica Charantia L.)

This study used Binjing II, Changbai and Biyu that are planted widely in Chongqing as materials to do tissue culture. We have built up tissue culture system of balsam pear completely. Above on it, we successfully used colchicine to induce tetraploid of balsam pear with infinitive bud and they grew up. Finally, we did the comparing analysis on physiological and biochemical trait between diploid and tetraploid of balsam pear. The main contents include below:1. Choose and pretreat material.We finally chose Binjing II, Changbai and Biyu that are planted widely in Chongqing as typical materials to do tissue culture. Binjing II and Biyu are green peel types, but Changbai is white peel type. The seed capsule of balsam pear is so thick and hard that it is hard to absorb water. So we should deal with it away before burgeon. There is a layer of mucous membrane on embryo that blocks water to be absorbed and brings pollution easily. We should clean it. And then dip embryo in tepid water to improve germination rate and trimness.2. Build up suitable sterilization wayJavel water's sterilization effect was not good. When using 0.1% HgCl₂, we should control the sterilization time strictly. It could not sterilize drastically below 6 minutes. But seed will be poisoned and germination rate will be very low if it's up 8 minutes. So use 0.1% HgCl₂ to sterilize 6 to 8 minutes is a proper way.3. Germinate in pure conditionIn pure condition, the germination rate of Binjing II, Changbai and Biyu were respectively 66.7%, 73.3% and 100%. And Biyu's germination was trim and strong,. It should be a good material for tissue culture.4. Calli inducement and infinitive bud differentiation Calli was easy to be induced in substrate of MS+6-BA+NAA. Calli inducement rate was 100% and not sensitive to hormone chroma. Infinitive bud differentiated very fast. It could differentiat normally after 5 or 6 days. Faster ones could differentiat after 2 or 3 days. We have found out the substrates, be suit for hypocotyl, are MS+6-BA3～5mg/L+NAA0.5mg/L. But differentiation rate was low, the hypocotyl of it was average 9.37%. Average differentiation rate of Binjing II was 7.33%, 14.30% was the highest differentiation rate when putting in the substrate of MS+6-BA4mg/L+NAA0.5mg/L. Average differentiation rate of Changbai was 9.55%, 14.30% was the highest differentiation rate when putting in the substrate of MS+6-BA3mg/L+NAA0.5mg/L. Average differentiation rate of Biyu was 11.23%, 16.70% was the highest differentiation rate when putting in the substrate of MS+6-BA3mg/L+NAA0.5mg/L. Cotyledon differentiation result was bad.5. Multiplication and propagation cultureIn order to obtain lots of balsam pear bud, we could put infinitive bud, terminal bud of pureseedling and hypocotyl segment with lateral bud growing point into multiplication substrate(MS+6-BA3mg/L+NAA0.5mg/L) to propagate. Each explant could form 3 to 4 buds. Cut theminto stem segment with growing point and propagate again. So we propagate the balsam pear budfast.6. Tetraploid inducementCulture the balsam pear bud of Biyu into the tetraploid inducement substrate, then into the multiplication substrate, and then into the rootage substrate. Cut root tine growing point to press chromosome piece and then observe it by microscope. We found out that colchicine chroma is 0.2% and disposal time is 48 hours is the suitable way to induce balsam pear tetraploid. The highest inducement rate is 14.30%.7. Tetraploid identificationIn order to obtain pure tetraploid, we propagate the induced material many eras. After rootage culture, pressing chromosome piece and observing by microscope, we found out that we can obtain pure tetraploid after propagating four eras the materials dealt with colchicine.8. Root and transplantCulture tetraploid materials in rootage substrate(1/2MS+NAA0.2mg/L) and they all root. When plumule is 4 to 5 centimeters, exercise it and then transplant. 60% of them can survive.9. Comparing analysis on physiological and biochemical trait between diploid and tetraploid balsam pear Stem thick and plant height were tested after transplanting 30 days. Use these leaves of middle part to do Comparing analysis on physiological and biochemical trait between diploid and tetraploid balsam pear. The results showed that tetraploid's stem thick, plant height, soluble protein, soluble sugar, chlorophyl, carotenoid, malondialdehyde, proline, peroxidase, and superoxide dismutase are all better than diploid's. So we can see that physiological and biochemical enginery of tetraploid is better than diploid's.This study used new hormone combination to do balsam pear tissue culture, fast propagated balsam pear, perfected balsam pear tissue culture system, grounded stable academic base for balsam pear tissue culture and other researches. Base on it, we can successfully induce pure balsam pear tetraploid with colchicine, innovate balsam pear idioplasm resource, and supply middle material for other breeding work, such as breeding triploid balsam pear. We farther do comparing analysis on physiological and biochemical trait between diploid and tetraploid balsam pear, supply referenced gist for study on physiological and biochemical enginery of diploid and tetraploid balsam pear.