The Ocurrence Of Unreduced Male Gametes And Creation Of Sexual Polyploid Germplasm Resources In Pharmaceutical Dendrobium

Pharmaceutical Dendrobium is a traditional herb in China with broad market prospect.Polyploidization is an important breeding method of medicinal plants.There were some reports concerning the creation of asexual polyploids in pharmaceutical Dendrobium,however,there were no reports on the development of sexual polyploid by the way of 2n gamete till today.In this study,nine species or cultivars,six interspecific hybrid populations and two intraspecific hybrid populations of medicinal Dendrobium were employed to investigate on the occurrence and cytological mechanism of 2n male gamete,as well as crossing compatibility of the hybrids,and on the basis,the hybrids with high occurrence frequency of 2n male gamete were screened to create sexual polyploids.The main results were as follows:1.Unreduced male gametes were commonly occurred in the nine cultivars of medicinal Dendrobium,but the occurrence frequency of 2n male gamete was obviously different among various cultivars.The average frequency of 2n male gamete was highest（2.32%）in D.hercoglossum’Silver’,others were less than 1%.Besides,the average frequency of 2n male gamete was different among different variaties of D.officinale,and the highest one was in D.officinale’BJ’（0.33%）,the lowest one was in D.officinale’HSH’（0.07%）.Eight germplasm resources with more than 1%occurrence frequency were obtained,of which two was selected from D.officinale’GH’and D.aphyllum,respectively,and six from D.hercoglossum’Silver’.2.Unreduced male gametes were also commonly existed in the nine kinds of medicinal Dendrobium hybrids,while there were obviously differences on the occurrence frequency of 2n male gamete among different hybrids.In six interspecific hybrids,the average frequency of 2n male gamete was highest（5.52%）in the offspring of D.officinale’BJ’×D.aphyllum,followed by D.aromatic（Baishi）×D.loddigesii（2.40%）.Compared to the interspecific hybrids,the average frequency of 2n male gamete in two intraspecific hybrids of D.officinale was lower,representing 0.40%and 0.33%respectively,indicating that the production rate of 2n male gamete can be significantly increased through distant hybridization.Forty hybrids with the occurrence frequency of 2n male gamete being more than 2%were obtained.3.The stability of the frequency of 2n male gamete in three medicinal Dendrobium species and five hybrids was identified.The results showed that during two years,the frequency of 2n male gamete in D.aphyllum,D.loddigesii and four hybrids was stable,while that in D.officinale’BJ’and D.officinale’BJ’×D.officinale’HSH’was instable.The results demonstrated that the occurrence of 2n male gametes was not only controlled by heredity,but also affected by the environmental conditions.4.The meiosis of pollen mother cells in D.officinale’BJ’and D.officinale’BJ’×D.aphyllum was observed.The results demonstrated the production of 2n male gamete in parents and their hybrids was both in the form of dyad and triad.The production of 2n male gamete in parents was mainly with the form of triad,while the hybrid with high occurrence frequency of 2n gamete was mainly through the form of dyad.The formation of dyads was due to the asynchronous meiotic division of microspore mother cell,which resulted in the absence of meiosis I and normal division of meiosis II in a few microspore mother cells.The occurrence of the triad was due to the formation of tripolar spindle at meiosis II.5.The hybrid F₁generation of the medicinal Dendrobium had low self-cross compatibility and high cross compatibility.The fruit setting rate of self-cross in F₁generation was from 0%to 41.67%,while that of the cross ranged from 0%to 100%.All the generations of 19 self-cross and hybrid combinations could aseptically germinate,and seed germination rate of three combinations was above 80%.Nine combinations with high seed germination rate were selected for protocorm differentiation,rooting and strengthening experiments,and the results showed that the plant regeneration rates were all above 50%.6.Based on the methods of morphological observation and flow cytometry,one and ten accessions of possible sexual polyploid were respectively obtained from the test-tube seedlings of one self-cross and five sib-cross of F₁hybrids,which indicated that it’s feasible to create sexual polyploid through the way of 2n gamete.Through the above studies,the occurrence discipline of 2n male gamete and its cytological mechanism was preliminarily clarified,the reliable technological system of polyploid creation through 2n male gamete was established,eleven polyploids of medicinal Dendrobium were successfully developed.Those laid a solid foundation for the polyploid breeding in medicinal Dendrobium.