Studies On Induction Of Polyploid And Breeding Of Good New Variety In Mulberry

Mulberry is an important economical woody plant. Its leaf is the main diet for silkworm. Mulerry fruits are eaten fresh and are also used in marmalades, juices, liquors natural dyes and in the cosmetics industry. The plant has also been used medicinally. The black and red mulberry fruits are healthier for human body because of high nutrient value and medical function. So the mulberry variety for fruit shows a good prosperity in productional application. Mulberry polyploid, especially triploids is welcomed because they have high yield, high-quality, disease-resistant, resists the going against strong, fine properties that adaptability is wide,etc. Breeding of artificial triploid in mulberry has become an important approach at home and abroad. Desirable triploids are being developed using induced superior tetraploids for hybridization with diploids. Most of natural tetriplods are unsuitable for superior tetraploid parents due to poor nutritional value and low propagation efficiency. So we have to get by artificial creation. At present, the main methods artificial tetraploid are Co-γradiation-induced mutation, Colchicine-induced mutation, crossbreeding in the field and are affected by environment easily. Most of the tetraploids by artificial diploid hybrid doubled have high yield and applied to the production. It is still an important way for polyploid breeding in mulberry through crossing the tetraploid with diploid and breeding triploid hybrid combinations by using induced tetraploid. So this study contains two sections: one section is in vitro induction of tetraploid in mulberry, the other section is selective breeding of artificial tetraploid with high leaf-yields and high fruit-yieds by Colchicine-induced in mulberry and a comparative study on morphological, anatomical Structure of stem and leaf, physiological and biochemical mechanism difference between tetraploid and diploid of mulberry. The main results were briefly summarized as follows:1. Establishment of in Vitro and Plant Regeneration System of MulberryThe aim of this study is to establish a rapid and efficient plant regeneration system ofmulberry through mulberry seed embryo. The seeds were collected during April-May every year from mature tree of Zhongsang5801×Naxi (Moms multicaulis Perr.) and preserved in 4℃refrigerator for providing explants at any time throughout the year. Seeds were washed and soaked for one day, and then their surfaces were sterilized in 70% alcohol for 1 min followed by 0.1% mercuric chloride for 8 min. Finally, the seeds were rinsed for 4-5 times in sterile distilled water for 1 min . The sterilized seeds were cultured in sterile distilled water on the rocking bed or placed on the moist filter paper in sterile Petri dishes. Seeds were germinated at 25±2℃using a 12 h photoperiod with a light intensity of 50μmol m-2 s-1 with fluorescent lights (40 W GE Deluxe Daylight bulbs). After 5 days, under aseptic conditions, germinated seedlings were cut with sharp scissors to produce segments of hypocotyls and cotyledons, the hypocotyl and cotyledonof every seedling was cut into three segments with 1.5mm long as explants. The result as follows: the optimum shoot inducing medium is MS+6-BA 3.0mg/L+IAA 0.3 mg/L+ dextrose 30 g/L + agar powder 0.4%. the optimum root inducing medium is MS+IBA0.5 mg/L. Addition of AgNO₃ to the medium promoted the differentiation percentage of (adventitious) shoots. shoot induction frequences of three segments of hypocotyl showed significant difference in the same media. The highest was the segment near the embryo bud 78. 7±2.1%, and the lowest was the segment near the embryo root 16±2.2%. The middle is 39.5±7.9%. the rooting rate was above 99.0% and the transplanting survival rate was above 95%.2. In Vitro Induction of Tetraploid in MulberryThe aim of this study is induction of tetraploid in mulberry in vitro. Seed, hypocotyl, cotyledon and multiple shoot was tested as explants. This paper designed 4 experiments: (1) seeds of crossbreed variety were treated with three different concentrations (0.15, 0.2 and 0.25%) of colchicine for 24h, 48h, 96h. (2) cotyledons were treated with three different concentrations (0.05, 0.1 and 0.2%) of colchicine for 20min, 40min, 60min. (3) multiple shoots(2-4cm) were treated with three different concentrations (0.1, 0.15 and 0.2%) of colchicine for 2 days or 3 days by impregnation (4) By using dropping liquid, multiple shoots(2-4cm) were treated with three different concentrations (0.5, 0.2and 0.25%) of colchicine for 5 days, twice every day. The result shows: tetraploidy plantlet induced only in multiple shoots; The combination of concentration of colchicine and explants might be the key factor. Tetraploidy at a frequency of 14% was obtained using 0.2% colchicine for 2d by impregnation, And the frequency of tetraploidy was 20% when 0.2% colchicine for 5d by using dropping liquid. The leaves of tetraploid that developed from colchicine treated buds were distorted while the upper newly formed leaves were large, thick and dark green, with stronger venation and deeper serration. The length and breadth of the leaves were also greater in colchicine-induced plants (20.4±0.9 cm and 17.1±0.1 cm) compared with that of untreated plants (16.7±1.1 cm and 12.8±1.7 cm). The tetraploids can be used not only as tetraploid parents but also as practical variety. This is the first report on in vitro induction of tetraploid in mulberry in China. Thus, it provides a new and efficient path for mulberry polyploid breeding.3. Induction of Tetraploid in Mulberry for Fruit and LeafThis study is in order to meet peasant's demand, increase income per hm² in economic benefit to develop secriculture industry. The objective is to select and breed new variety with not only high leaf-yields but also high fruit-yields in mulberry. Based on long term surveying on the local mulberry resources. Zhongsang 5801 is the better diploidy parent, Its leaf is bigger and good quality and its fruit has a nice taste. We injected colchicine solution into winter bud in mulberry for 8d by the concentration of 2ppm6-BA+0.2%,2ppm6-BA+0.22%,2ppm6-BA+0.25% on Feb 22,2005 After 1 month, we identified and separated 1 tetraploid bud from 277 injected buds and propagated the tetraploid by grafting and matched cultivation managemen in Dec,2005. The tetraploid plant of muberry showed better economic properties than the diploidy parents. The mean leaf yield reached a higher level to 2088kg/667m² and fruit yield reached 777. 5kg/667m² in Chongqing regional test from in 2008, 34.7% in leaf and 46.57% in fruit higher than that of the control variety "Hongguo 2" in 2008; So this way will increase the income of farmers and strengthen their enthusiasm of planting mulberry to raise silkworm. It leads to sustainable development of sericulture.4. Comparison of Growth and Physiological Indices between Diploid and Tetraploid Mulberry PlantsThe comparative study of growth and physiological indices between diploid and tetraploid mulberry plants were carried out. The difference were evaluated for leaf character, leaf yield, fruit yield, chemical composition, soluble protein content, total sugar content; Amino acid, Vc, polyphenol, anthocyanin and flavone of fruit and the activity of POD, SOD. The results have shown statistically significant diferences between tetraploid and diploids for all the parameters. mulberry leaf yield increased 19.05% and the mulberry fruit yield increased 16.1% of titraploid than of diploid ; the content of prtain and soluble sugar in the mulberry leaves was 10.2% and 10.7% higher than diploid; The anti-oxidative substance such as Vc, polyphenol, anthocyanin and flavone in tetraploid were 12.9%, 43.3%, 34.1%, 47.6% higher than in diploid. The activity of POD, SOD in tetaploid were significantly higher than that in diploid . So we speculated that Tetraploids induced from hybrid seedling diploid were much stronger growing and stronger stress resistance. The researches provided some useful information of polyploid breeding.5. Photosynthetic Physiological Characteristics and Photosynthetic Structure of Tetraploid and DiploidThe aim of this study is to elucidate the physiological mechanism of stronger growing in tetraploid than that in diploid. Under field cultivation conditions, Photosynthetic pigment (chlorophll-a, chlorophll-b) contents per unit area leaf of mulberry increased with increasing ploidy. There was significant difference between tetraploid and diploid. Net photosynthetic rate (Pn) of different ploidy watermelon showed(?)midday depression' phenomenon in April. The Pn diurnal changes varied as atwo-peaked curve, its values reached at 11:00 am. and 16:00 pm.; The Pn of first peak was higher than the second. The Pn was 4x＞2x . The critical points of light saturation and maximum photosynthetic rate, apparent quantum yield (AQY), carboxylation efficiency is 329umol·m^-2·s^-1, 21.4μmol·m^-2·s^-1, 0.069, 0.096 respectively in tetraploid and 339 umol·m^-2·s^-1, 15.7μmol·m^-2·s^-1, 0.05, 0.067 respectively in diploid. The tetraploid shows significant superior photosynthetic character. The abilitly of carboxylation of tetraploid was one time higher than diploid, which may be closely related to net photosynthetic rate.Observation of the morphology and anatomy of tetraploid showed that leaf palisade and sponge tissue thickened, the stoma become larger, the number of chloroplast in per guard cell increased. Convex lens observed that the number of starch grain in each cell reduced in tetraploid and the number of basal granules and basal granule layers of leaf of grape in greenhouse were higher than diploid. These basal granule layers are highly ordered and uniform to improve the photosynthetic rate. Forthermore, structure of conducting tissue was studied and the number of vessel increased and the vessel in tetraploid was bigger than diploid. These structural change constitute the physiological mechanism of improvement of yield in tetraploid.